Ligase fusion proteins and application thereof

ABSTRACT

The present disclosure relates to the field of biotechnology. In particular, provided are a ligase fusion protein and an immobilized ligase comprising the same. Also provided is use of the ligase fusion protein or the immobilized ligase in the preparation of conjugates. Further provided is a process for the preparation of conjugates using a ligase or a ligase unit.

TECHNICAL FIELD

The present disclosure relates to the field of biotechnology, inparticular to a ligase fusion protein and an immobilized ligasecomprising the same. Also provided is use of the ligase fusion proteinor the immobilized ligase in the preparation of conjugates. Furtherprovided is a process for the preparation of conjugates using a ligaseor a ligase unit.

BACKGROUND

Demands for high-quality conjugates, especially bioconjugates, such asthose for bioscience research, diagnosis or therapeutics purposes, areincreasing rapidly. However, the high-throughput production ofbioconjugates is far from satisfying, partially because that the complexnature of biomolecules makes the high-quality standards forbioconjugates difficult to be met.

Conventional conjugation process is chemistry based. For example, in atypical process for antibody-drug conjugate (ADC) production, the drugis chemically conjugated to lysine or cysteine residue in the antibodyvia a linker. The antibody is prepared through upstream and downstreampurification processes before entering the conjugation process. Afterthe conjugation step, another downstream purification process isrequired to remove the aggregates, solvents, by-products and impuritiesfrom the ADC. The multiple downstream steps along the process fromantibody preparation to ADC production significantly increase cost andtime, and simultaneously lower the yield. Moreover, the conjugationreaction has to be conducted in chemical isolators for safety reasons,making the process difficult to scale up. Overall, the conventionalprocesses involve multiple upstream and downstream purification steps,which are time-consuming, uneconomic, inflexible and lack scalability.

Ligases, such as Sortase enzymes are applied to catalyze conjugation ina highly substrate-specific and efficient manner under mild conditions(e.g., WO2015/165413A1, WO2014/177042 and WO2014/140317), which mayreduce time, cost, and waste. Despite the many advantages, however,industrial application of ligases for conjugation is still limited dueto several challenges.

Challenges such as low operational stability and reusability of theenzymes may be somewhat overcome by enzyme immobilization. Immobilizedsortase A on cyanogen-bromide activated Sepharose (see, e.g., Witte etal., Site-specific protein modification using immobilized sortase inbatch and continuous-flow systems, Nat Protoc, (2015), 10(3): 508-516)or His₆-tagged sortase A immobilized on nickel-modified magneticparticles (see, e.g., Zhao et al., One-step purification andimmobilization of extracellularly expressed sortase A by magneticparticles to develop a robust and recyclable biocatalyst, Sci Rep,(2017), 7: 6561) has been employed for conjugation.

However, removal of residual enzyme contaminants carried over from theupstream catalytic reaction remains a major concern for mostenzyme-catalyzed conjugates, especially for bioconjugates, becauseresidual enzyme contaminants (in the case of immobilized enzyme, freeenzymes non-specifically adsorbed on the support may still fall off) canbe difficult to remove. Therefore, there is a need for ligases that arecost efficient, stable, controllable and easily removable from theconjugate product.

SUMMARY

In a general aspect, provided is a ligase fusion protein comprising aligase and a Halo tag.

In some embodiments, the ligase is a transpeptidase. In someembodiments, the ligase is a sortase. In some embodiments, the ligase isa sortase A. In some preferred embodiments, the sortase A comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:1-26 or an amino acid sequence having a sequence identity of at leastabout 85%, at least about 90%, at least about 95%, or at least about 99%thereto. In some other preferred embodiments, the sortase A comprisesamino acid substitutions of SNAT, YNAT, WNDT or VNNS at positions 34,100, 105 and 136, preferably, the sortase A comprises the amino acidsequence of SEQ ID NO: 27 or an amino acid sequence having a sequenceidentity of at least about 85%, at least about 90%, at least about 95%,or at least about 99% thereto.

In an embodiment, the Halo tag is a mutant haloalkane dehalogenase or avariant thereof that removes the halogen from a haloalkyl substrate andforms a covalent linkage with the remaining alkyl group. In someembodiments, the Halo tag comprises the amino acid sequence of SEQ IDNO: 28 or an amino acid sequence having a sequence identity of at leastabout 85%, at least about 90%, at least about 95%, or at least about 99%thereto.

In a preferable aspect, provided is a ligase fusion protein having analtered isoelectric point (pI) compared to the ligase from which it isderived, wherein the ligase has an alkaline pI and the Halo tag has anacidic pI. In some embodiments, the ligase has an isoelectric point (pI)of about 7.5 to about 10.0, the Halo tag has an isoelectric point ofabout 4.5 to about 5.0, and the pI of the ligase fusion protein is about2.0 to about 4.5 pH units lower than that of the ligase.

In another general aspect, provided is an immobilized ligase, comprisingthe ligase fusion protein according to the present disclosureimmobilized to a support.

Also provided is use of the ligase fusion protein or the immobilizedligase according to the present disclosure in the preparation of aconjugate.

In yet another general aspect, provided is a process for the preparationof a conjugate comprising a first moiety and a second moiety, comprisingthe steps of:

(a) providing System 1 comprising the first moiety and providing System2 comprising the second moiety; and

(b) contacting a ligase unit with System 1 and System 2 in step (a) tocatalyze the conjugation reaction between the first moiety and secondmoiety to obtain the conjugate,

wherein the ligase unit comprises a ligase,

the first moiety and the second moiety each independently comprises abiomolecule, a protein, an antibody, an antibody fragment, a receptor, asignal transduction factor, a cell growth factor, a nucleic acid or anucleic acid analogue, a small molecule compound, a glycan, a PEGmoiety, a radionuclide, a cytokine, an immunomodulator, a tracermolecule, a fluorophore, a fluorescent molecule, a peptide, apolypeptide, or a peptidomimetic; and

one of the first moiety and the second moiety further comprises therecognition motif of the ligase donor substrate, and the other one ofthe first moiety and the second moiety comprises the recognition motifof the ligase acceptor substrate.

In some embodiments, the ligase unit comprises a free ligase, preferablya transpeptidase, more preferably a sortase, even more preferably asortase A, most preferably the ligase unit comprises the ligase fusionprotein according to the present disclosure.

In some other embodiments, the ligase unit comprises a ligaseimmobilized to a support, preferably, the ligase is covalentlyimmobilized to the support, preferably the ligase is a transpeptidase,more preferably a sortase, even more preferably a sortase A, mostpreferably the ligase unit comprises the immobilized ligase according tothe present disclosure.

In some embodiments, at least one of System 1 and System 2 in step (a)comprises one or more impurities. In some other embodiments, at leastone of System 1 and System 2 in step (a) is a harvested clarified cellculture fluid (HCCF).

In some embodiments, the process further comprises the steps of

(1) subjecting System 1 in step (a) before step (b), and/or

(2) subjecting System 2 in step (a) before step (b), and/or

(3) subjecting the conjugate obtained in step (b),

to one or more chromatography steps to remove one or more impurities.

The chromatography step can be independently selected from the groupconsisting of affinity chromatography, hydrophobic interactionchromatography, ion exchange chromatography, mixed mode chromatography,hydroxyapatite chromatography and a combination thereof. Preferably, thechromatography step is selected from affinity chromatography, ionexchange chromatography, and a combination thereof.

In some embodiments, at least one of the first moiety and the secondmoiety comprises an antibody or an antibody fragment, and at least oneof steps (1)-(3) comprises an affinity chromatography; preferably, theantibody or antibody fragment comprises an Fc fragment, and the affinitychromatography is Protein A affinity chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the sortase activities of an exemplary SrtA derived fromStaphylococcus warneri (SEQ ID NO: 3) and its SNAT variant.

FIG. 2 depicts the activities of purified (A) Halo-Sortase, (B)His₆-Sortase and (C) GB1-Sortase.

FIG. 3 depicts enzyme capacities of different Chloro Resins.

FIG. 4 depicts catalytic activities (expressed as DAR) of immobilizedHalo-Sortase prepared from different Chloro Resins.

FIG. 5 shows the solubility of ADC products catalyzed by GB1-Sortase,His-Sortase or Halo-Sortase at low temperature.

FIG. 6 depicts chromatography profiles of (A) ADC, (B) Halo-Sortase and(C) ADC+Halo-Sortase on AEX using Q Sepharose FF medium.

FIG. 7 depicts chromatography profiles of (A) ADC and (B) Halo-Sortaseon CEX using Capto S impact medium.

FIG. 8 depicts the DAR compositions of conjugates comprised in the crudeconjugate mixture of Process 2 analyzed by HIC-HPLC.

FIG. 9 shows the amount of residual impurities in samples containing thetarget ADCs after each chromatography step in Process 2: Protein A, mAbeluate from Protein A affinity chromatography; AEX, ADC flow-throughfrom AEX; CEX, ADC eluate from CEX.

FIG. 10 depicts the DAR compositions of conjugates comprised in thecrude conjugate mixture of Process 1 analyzed by HIC-HPLC.

FIG. 11 shows the amount of residual impurities in samples containingthe target ADCs after each chromatography step in Process 1: Protein A,ADC eluate from Protein A affinity chromatography; AEX, ADC flow-throughfrom AEX; CEX, ADC eluate from CEX.

FIG. 12 shows the amount of residual impurities in samples containingthe target ADCs after each chromatography step in Process 3: 1st ProteinA, mAb eluate from Protein A affinity chromatography; 2nd Protein A, ADCeluate from Protein A affinity chromatography; AEX, ADC flow-throughfrom AEX; CEX, ADC eluate from CEX.

FIG. 13 depicts amounts of residual Halo-Sortase in samples containingthe target ADCs: Conjugation, crude conjugate mixture collected as theflow-through from the Halo-Sortase column; Protein A, ADC eluate fromProtein A affinity chromatography; AEX, ADC flow-through from AEX; CEX,ADC eluate from CEX.

FIG. 14 shows optimized chromatography profiles of Linker-Toxin(linker-payload intermediate) removal by (A) Protein A media fromBiomax, (B) Protein A media from GE; (C) CEX media from GE.

FIG. 15 shows flowcharts illustrating the steps of ADC preparation usingconventional process (Conventional ADC process) and the processesaccording to the present disclosure (ADC Process 1, ADC Process 2, ADCProcess 3 and ADC Process 4): Protein A, Protein A chromatography; lowpH, low-pH treatment; UF/DF, ultrafiltration/diafiltration; AEX, anionexchange chromatography; CEX, cation exchange chromatography; HIC,hydrophobic interaction chromatography; Mab DS, monoclonal antibodydownstream processes.

DETAILED DESCRIPTION General Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart. In addition, the terms and experimental procedures relating toprotein and nucleic acid chemistry, molecular biology, cell and tissueculture, microbiology and immunology are those terms and commonprocedures widely used in the art. When a trade name is present herein,it refers to the corresponding commodity or the active ingredientthereof. All patents, published patents applications and publicationscited herein are hereby incorporated by reference. Meanwhile, for betterunderstanding of the present disclosure, definitions and explanations ofrelevant terms are provided below.

As used herein, the expression “at least one” or “one or more” refers toone, two, three, four, five, six, seven, eight, nine or more, onehundred, two hundred, three hundred, four hundred, five hundred, sixhundred, seven hundred, eight hundred, nine hundred or more, etc. Asused herein, “a” and “an” unless clearly indicated to the contrary,should be understood to mean “at least one”.

When a certain amount, concentration, or other value or parameter is setforth in the form of a range, a preferred range, or a preferred upperlimit or a preferred lower limit, it should be understood that it isequivalent to specifically revealing any range formed by combining anyupper limit or preferred value with any lower limit or preferred value,regardless of whether the said range is explicitly recited. Unlessotherwise stated, the numerical ranges listed herein are intended toinclude the endpoints of the range and all integers and fractions(decimals) within the range. For example, the expression “i” is aninteger of 2 to 20″ means that i is any integer of 2 to 20, for example,i can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20. Other similar expressions should also be understood in a similarmanner.

The terms “about” and “approximately”, when used in connection with anumerical variable, such as a concentration, an isoelectric point (pI),a pH, a temperature or a certain range, generally mean that the value ofthe variable and all values of the variable are within experimentalerror (for example, within a 95% confidence interval for the mean) orwithin ±10% of a specified value, or a wider range.

The term “optional” or “optionally” means the event described subsequentthereto may, but not necessarily happen, and the description includesthe cases wherein the said event or circumstance happens or does nothappen.

The expression “comprising” or similar expressions “including”,“containing” and “having” are open-ended, and do not exclude additionalunrecited elements, steps, or ingredients. The expression “consistingof” excludes any element, step, or ingredient not designated. Theexpression “consisting essentially of” means that the scope is limitedto the designated elements, steps or ingredients, plus elements, stepsor ingredients that are optionally present that do not substantiallyaffect the essential and novel characteristics of the claimed subjectmatter. It should be understood that the expression “comprising”encompasses the expressions “consisting essentially of” and “consistingof”.

As used here, the definition of “biomolecule” encompasses proteins,nucleic acids, lipids, carbohydrates, small nucleotides, amino acids andderivatives thereof.

As used herein, a “nucleic acid” or a “polynucleotide” refers to apolymer of at least two nucleotides or nucleotide derivatives joinedtogether by phosphodiester bonds, including deoxyribonucleic acid (DNA)and ribonucleic acid (RNA).

As used herein, a “vector” is a vehicle used to transfer exogenousnucleic acids into a host cell, where the exogenous nucleic acids areamplified or expressed. As used herein, the definition of “vector”encompasses plasmids, linearized plasmids, viral vectors, cosmids, phagevectors, phagemids, artificial chromosomes (e.g., yeast artificialchromosomes and mammalian artificial chromosomes), etc. As used herein,a vector could be expressible and/or replicable inside a host cell,meaning that the vector is able to express RNA polynucleotides orpolypeptides and/or to produce multiple copies of the vector in the hostcell. To be “expressible” or “replicable”, a vector could comprisenucleic acid sequences or elements operably linked to a promoter. Asused herein, “operably linked” with reference to nucleic acid sequencesor elements means that these nucleic acid sequences are functionallyrelated to each other. For example, a promoter can be operably linked toa nucleic acid sequence encoding a polypeptide, whereby the promoterregulates or mediates the transcription of the nucleic acid. Thoseskilled in the art could select and use appropriate vectors for aparticular purpose.

As used herein, “peptide”, “polypeptide” or “protein” refers to two ormore amino acids covalently joined. Unless otherwise specified, theseterms are interchangeably.

As used herein, “sequence identity” has an art-recognized meaning andthe percent of sequence identity between two polypeptides can becalculated by aligning the two sequences using publicly availablealgorithms, such as the Basic Local Alignment Search Tool (BLAST) andthe Fast Adaptive Shrinkage/Thresholding Algorithm (FASTA) (see, e.g.:Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994). While there are a number of methods to measureidentity between two polypeptides, the term “identity” is well known toskilled artisans (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073(1988)).

As used herein, the term “variant” refers to a protein havingsubstitutions, deletions, insertions of one or more residues whencompared to a reference protein. The reference protein can be anaturally occurring protein that can be isolated from natural source(i.e., a wild-type protein) or an engineered protein. As used herein,the function or activity of a variant, such as a sortase A variant or aHalo tag variant, is substantially similar with or comparable to orhigher than that of the reference sortase A or Halo tag, respectively.

In the context of the present specification, the positions of aminoacids in a protein are defined as follows: (i) starting from theN-terminus; and (ii) the position of the 1^(st) amino acid from theN-terminus is designated as 1. An amino acid (such as Ser) at a givenposition (such as position 34) can be expressed as Ser34. An amino acid(such as His) at a given amino acid position (such as position 272)substituted with another amino acid (such as Phe) can be expressed asHis272Phe.

As used herein, a “ligase” refers to an enzyme that can catalyze thecovalent linkage of two or more molecules. A ligase can specificallycatalyze the conjugation between a first moiety comprising therecognition motif of the ligase donor substrate and a second moietycomprising the recognition motif of the ligase acceptor substrate toproduce a target conjugate.

As used here, the term “transpeptidation reaction” refers to a chemicalreaction in which one or more amino acids (such as a peptide) istransferred from one molecule to another molecule. A transpeptidase isan enzyme that is able to catalyze a transpeptidation reaction between adonor substrate and an acceptor substrate. In a simplifiedtranspeptidation reaction catalyzed by a sortase, the sortase firstcleaves the recognition motif of a ligase donor substrate (also referredto as a donor recognition motif, such as LPXTG, when SrtA is used) toform a substrate-enzyme intermediate through formation of a thioesterbond; next, the recognition motif of a ligase acceptor substrate (alsoreferred to as an acceptor recognition motif, such as GGG)nucleophilically attacks the thioester bond to release the enzyme andform a new peptide bond between the two substrates. A transpeptidationreaction often results in conjugation of two parties to form aconjugate.

As used herein, the term “conjugation” refers to the covalent linkage ofat least two parties (e.g., at least two molecules or at least two endsof the same molecule).

As used herein, a “conjugate” can be prepared from at least two parties(e.g., at least two molecules or at least two ends/side chains of thesame molecule) through covalent linkage.

As used herein, a “bioconjugate” refers to a conjugate with at least oneof the conjugated parties being a biomolecule. Examples of bioconjugatesinclude therapeutic molecules conjugated to polymer, lipid, antibody,peptide, aptamer, or small molecular ligands, such as siRNA conjugates,peptide hormone conjugates, peptide-peptide conjugates, peptide-drugconjugates, antibody-drug conjugates and multispecific antibodies, orthe like.

The term “targeting molecule” refers to a molecule that has an affinityfor a particular target (e.g., receptor, cell surface protein, cytokine,etc.). A targeting molecule can deliver the payload to a specific sitein vivo through targeted delivery. A targeting molecule can recognizeone or more targets. The specific target sites are defined by thetargets it recognizes. For example, a targeting molecule that targets areceptor can deliver a cytotoxin to a site containing a large number ofthe receptor. Examples of targeting molecules include, but are notlimited to antibodies, antibody fragments, binding proteins for a givenantigen, antibody mimics, scaffold proteins having affinity for a giventarget, ligands, and the like.

As used herein, the term “antibody-drug conjugate (ADC)” refers to aconjugate comprising an antibody or an antibody fragment coupled to apayload covalently.

As used herein, the terms “activity”, “enzymatic activity” and“catalytic activity” of a ligase (such as a sortase) refer to theability of the ligase to catalyze a conjugation reaction and can be usedinterchangeably. As used herein, the catalytic activity of a sortase ina conjugation reaction, for example, a conjugation reaction between anantibody and a payload, can be expressed as conjugation efficiency(conjugation efficiency=(molars of conjugated antibody: molars of totalantibody)×100%) or DAR (Drug-to-Antibody Ratio, average drug to antibodyratio for a given preparation of antibody drug conjugate) distribution.

As used herein, the term “antibody (Ab)” is an immunoglobulin (Ig)molecule or a derivative thereof that specifically binds to an antigenthrough at least one antigen-binding site. A “conventional” or“full-length” antibody typically consists of four polypeptides: twoheavy chains (HC) and two light chains (LC). As used herein, thedefinition of “antibody” encompasses conventional antibodies,recombinant antibodies, multispecific antibodies (e.g., bispecificantibodies), fully human antibodies, non-human antibodies, humanizedantibodies, chimeric antibodies, intrabodies, diabodies, nanobodies(i.e., single-domain antibodies, VHH domains), and anti-idiotypicantibodies. Also contemplated are members of any immunoglobulin type(e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g. IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass (e.g., IgG2a and IgG2b), or anyderivatives thereof.

As used herein, an “antibody fragment” of an antibody refers to anyportion of an antibody comprising fewer amino acid residues than afull-length antibody, such as an antigen-binding fragment that containsat least a portion of the variable domains (e.g. one or more CDRs) ofthe antibody and specifically binds to the same cognate antigen as thefull-length antibody, or an Fc fragment that contains heavy chainconstant regions of the antibody and binds to Fc receptors on the cellsurface. Antibody fragments can be obtained by various methods, such aschemical or enzymatic treatment, chemical synthesis or recombinant DNAtechnology. Examples of antibody fragments include, but are not limitedto, Fv (the fragment variable region), scFv (single-chain Fv fragment),dsFv (disulfide-stabilized variable fragment), scdsFv (single-chaindisulfide-stabilized variable fragment), diabody, Fd (the fragmentdifficult), Fab (the fragment antigen binding), scFab (single-chainFab), Fab′, F(ab′)₂, Fc (the fragment crystallizable region) and anyderivatives thereof.

As used herein, the term “payload” refers to a functional moiety whichis comprised in a conjugate, for example, linked via a linker. Examplesof payload include, but are not limited to, small molecule compounds(also referred to as small molecule drugs, e.g., inhibitors and toxins(such as cytotoxins)), radionuclides (e.g., ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi,⁶⁷Ga, ¹²³I, ¹²⁴I, 125I, ¹³¹I, ¹¹¹In, ¹⁷⁷Lu, ^(191m)Pt, ^(195m)Pt, ¹⁸⁶Re,¹⁸⁸Re, ¹¹⁹Sb, ¹⁵³Sm, ^(99m)Tc, ²²⁷Th and ⁹⁰Y), glycans, PEG moieties,nucleic acids and analogues (e.g., interfering RNAs), tracer molecules(e.g., fluorophores and fluorescent molecules), polypeptides (e.g.,protein tags, bioactive peptides, enzymes, antibodies and antibodyfragments, and protein toxins) and peptidomimetics. As used herein,payloads comprising a linker (such as a linker comprising a recognitionmotif of the ligase substrate) and a payload as described above areconsidered.

As used herein, the term “natural amino acid” refers to an amino acidthat is a protein constituent amino acid, including the common twentyamino acids (alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine), and the less common selenocysteine andpyrrolysine.

As used here, the term “unnatural amino acid” refers to an amino acidthat is not a protein constituent amino acid. In particular, the termrefers to an amino acid that is not a natural amino acid as definedabove.

As used herein, the term “peptidomimetic” refers to a compound thatmimics the conformation and desirable features of a particular peptide.

As used herein, a “receptor” refers to a structure inside or on thesurface of a cell that binds a specific substance and causes a specificeffect in the cell. Receptors may include T-cell receptors, B-cellreceptors, and receptors of signaling molecules, cell growth factors andcytokines as described herein.

As used herein, a “signal transduction factor” refers to any substancethat plays a role in a signal transduction event across or through acell. Signal transduction factors may include, but not limited to,signaling molecules (such as steroid hormones, retinoic acid, thyroidhormone, vitamin D₃, peptide hormones, neuropeptides, eicosanoids,neurotransmitters and cytokines) and receptors thereto.

As used herein, the term “immunomodulator” refers to a biologicallyactive substance that is capable of affecting the functioning of theimmune system. An immunomodulator can be immunosuppressive (such as animmunosuppressant/immunosuppressive agent) or immunostimulatory (such asan immunostimulant/immunostimulator). Examples of immunomodulators mayinclude, but are not limited to, cytokines, thymus hormones (e.g.,thymulin, thymosin and thymopoietin), lentinan, β-glucans, inulin,levamisole, isoprinosine, IMPDH inhibitors (e.g., azathioprine,leflunomide, mycophenolic acid, mizoribine, ribavirin, and tiazofurin),calcineurin inhibitors (e.g., cyclosporine and tacrolimus), mTORinhibitors (e.g., sirolimus and everolimus), P38 inhibitors, NF-κBinhibitors (e.g., bortezomib), corticosteroids (e.g., prednisone,budesonide and prednisolone), Janus kinase inhibitors (e.g., tofacitiniband baricitinib), anti-cytokine antibodies and antibodies against T-cellreceptors.

As used herein, the term “cell growth factor” refers to any substancethat is capable of stimulating cellular growth, healing, proliferation,survival and differentiation. Examples of growth factors may include,but are not limited to, epidermal growth factor (EGF), fibrablast growthfactor (FGF), transforming growth factor (TGF), platelet-derived growthfactor (PDGF), teratocarcinoma-derived growth factor (TDGF),insulin-like growth factor (IGF), nerve growth factor (NGF), vascularendothelial growth factor (VEGF) and erythropoietin (EPO).

As used herein, the term “cytokine” refers to any substance released bythe cells of the immune system and having an effect on other cells.Examples of cytokines may include chemokines, lymphokines,colony-stimulating factors (CSFs), monocyte chemoattractant proteins(MCPs), angiogenesis factors, interleukins, interferons, tumor necrosisfactors (TNFs), growth factors, and other secreted and cell surfacemolecules that transmit signals to other cells. Cytokines include, butare not limited to, INFα, INFβ, INFγ, IL-1, IL-2, IL-4 IL-6, IL-8/CXCL8IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-23, IP-10/CXCL10,eotaxin/CCL11, MCP-1/CCL2, MIP-1a/CCL4, RANTES/CCL5, TNFα, TNFβ, andgrowth factors.

A small molecule compound refers to a molecule with a size comparable tothat of an organic molecule commonly used in medicine. The term does notencompass biological macromolecules (e.g., proteins, nucleic acids,etc.), but encompasses low molecular weight peptides or derivativesthereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides,and the like. Typically, the molecular weight of the small moleculecompound can be, for example, about 100 to about 2000 Da, about 200 toabout 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da,about 200 to about 700 Da, about 200 to about 600 Da, about 200 to about500 Da. As used herein, a small molecule compound may also be known as adrug.

Cytotoxin refers to a substance that inhibits or prevents the expressionactivity of a cell, cellular function, and/or causes destruction ofcells. In some cases, the cytotoxins currently used in ADCs may be moretoxic than commonly used chemotherapeutic drugs. Examples of cytotoxinsinclude, but are not limited to, drugs that target the followingtargets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated proteintransport, regulation of apoptosis. The drug that targets microtubulecytoskeleton may be, for example, a microtubule-stabilizing agent or atubulin polymerization inhibitor. Examples of microtubule-stabilizingagents include but are not limited to taxanes. Examples of tubulinpolymerization inhibitors include but are not limited to maytansinoids,auristatins, vinblastines, colchicines, and dolastatins. TheDNA-targeting drug can be, for example, a drug that directly disruptsthe DNA structure or a topoisomerase inhibitor. Examples of drugs thatdirectly disrupt DNA structure include but are not limited to DNA doublestrand breakers, DNA alkylating agents, DNA intercalators. The DNAdouble strand breakers can be, for example, an enediyne antibiotic,including but not limited to dynemicin, esperamicin, neocarzinostatin,uncialamycin, and the like. The DNA alkylating agent may be, forexample, a DNA bis-alkylator (i.e. DNA-cross linker) or a DNAmono-alkylator. Examples of DNA alkylating agents include but are notlimited to pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer,1-(chloromethyl)-2,3-dihydrogen-1H-benzo[e]indole (CBI) dimer, CBI-PBDheterodimer, dihydroindolobenzodiazepine (IGN) dimer, duocarmycin-likecompound, and the like. Examples of topoisomerase inhibitors include butare not limited to camptothecins and anthracyclines. The RNA-targetingdrug may be, for example, a drug that inhibits splicing, and examplesthereof include but are not limited to pladienolide. Drugs that targetkinesin-mediated protein transport can be, for example, mitotic kinesininhibitors including, but not limited to, kinesin spindle protein (KSP)inhibitors.

A spacer is a structure that is located between different structuralmodules and can spatially separate the structural modules. Thedefinition of spacer is not limited by whether it has a certain functionor whether it can be cleaved or degraded in vivo. Examples of spacersinclude but are not limited to amino acids and non-amino acidstructures, wherein non-amino acid structures can be, but are notlimited to, amino acid derivatives or analogues. “Spacer sequence”refers to an amino acid sequence serving as a spacer, and examplesthereof include but are not limited to a single amino acid such as Leu,Gln, etc., a sequence containing a plurality of amino acids, forexample, a sequence containing two amino acids such as GA, etc., or, forexample, GGGS, GGGGSGGGGS, etc. Other examples of spacers include, forexample, self-immolative spacers such as PAB (p-aminobenzyl), and thelike.

The term “alkyl” refers to a straight or branched saturated aliphatichydrocarbon group consisting of carbon atoms and hydrogen atoms, whichis connected to the rest of the molecule through a single bond. Thealkyl group may contain 1 to 20 carbon atoms, referring to C₁-C₂₀ alkylgroup, for example, C₁-C₄ alkyl group, C₁-C₃ alkyl group, C₁-C₂ alkyl,C₃ alkyl, C₄ alkyl, C₃-C₆ alkyl. Non-limiting examples of alkyl groupsinclude but are not limited to methyl, ethyl, propyl, butyl, pentyl,hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl,2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl,neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl,2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl,3,3-dimethylbutyl, 2,2-dimethyl butyl, 1,1-dimethylbutyl,2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or theirisomers. A bivalent radical refers to a group obtained from thecorresponding monovalent radical by removing one hydrogen atom from acarbon atom with free valence electron(s). A bivalent radical have twoconnecting sites which are connected to the rest of the molecule. Forexample, an “alkylene” or an “alkylidene” refers to a saturated divalenthydrocarbon group, either straight or branched. Examples of alkylenegroups include but are not limited to methylene (—CH₂—), ethylene(—C₂H₄—), propylene (—C₃H₆—), butylene (—C₄H₈—), pentylene (—C₅H₁₀—),hexylene (—C₆H₁₂—), 1-methylethylene (—CH(CH₃)CH₂—), 2-methylethylene(—CH₂CH(CH₃)—), methylpropylene, ethylpropylene, and the like.

As used herein, when a group is combined with another group, theconnection of the groups may be linear or branched, provided that achemically stable structure is formed. The structure formed by such acombination can be connected to other moieties of the molecule via anysuitable atom in the structure, preferably via a designated chemicalbond. For example, when describing a combination of a C₁₋₄ alkylene withone of the groups including —CH₂—, —NH—, —(CO)—, —NH(CO)—, —(CO)NH—, theC₁₋₄ alkylene may form a linear connection with the above groups, suchas C₁₋₄ alkylene-CH₂—, C₁₋₄ alkylene-NH—, C₁₋₄ alkylene-(CO)—, C₁₋₄alkylene-NH(CO)—, C₁₋₄ alkylene-(CO)NH—, —CH₂—C₁₋₄ alkylene, —NH—C₁₋₄alkylene, —(CO)—C₁₋₄ alkylene, —NH(CO)—C₁₋₄ alkylene, —(CO)NH—C₁₋₄alkylene. The resulting bivalent structure can be further connected toother moieties of the molecule.

As used herein, the term “isoelectric point (pI)” is the pH (power ofhydrogen) value of an aqueous solution of a molecule (such as a protein)at which the molecule has no net surface charge and is expressed as pHunits. The pI of a protein can be experimentally measured using methodswell-known in the art, such as, imaged capillary isoelectric focusing(iCIEF) and capillary isoelectric focusing (CIEF). Differentbiomolecules (proteins, nucleic acids, polysaccharides, etc.) withdifferent pIs may be differently charged at a given pH, allowing them tobe separated by methods such as ion exchange chromatography orisoelectric focusing.

As used herein, a molecule having an “alkaline pI” means that the pI ofthe molecule is below 7.0. As used herein, a molecule having an “acidicpI” means that the pI of the molecule is above 7.0.

As used herein, a “protein tag” refers to a polypeptide that can beintroduced to a molecule of interest to facilitate the detection,isolation, immobilization or capture of the molecule of interest, orimprove one or more properties (such as expression level, solubility andstability) of the molecule of interest.

Ion exchange chromatography (IEX) separates biomolecules based ondifferences in their net surface charge and differences in theiraffinity towards the ion exchanger (also referred to as the medium,resin or stationary phase). It is a commonly used technique forbiomolecule purification. For example, in the anion exchangechromatography, a protein with a pI below the buffer pH will havenegative net surface charge and bind to the positively charged anionexchanger; however, another protein with a pI above the buffer pH willhave positive net surface charge and do not bind to the positivelycharged anion exchanger, and will thereby pass through the medium alongwith the buffer.

As used herein, the term “support” refers to a water-insoluble substancethat can be isolated from a reaction mixture in solid or semi-solidform, such as a surface, a gel, a polymer, a matrix, a particle, aresin, a bead or a membrane.

The term “clarification” refers to removal of insoluble impurities froma system containing a biomolecule of interest. The process of“clarification” could be monitored by reduced turbidity, as measured forexample, in nephelometric turbidity unit (NTU).

The term “polishing step” refers to a step to further remove minorcontaminants and aggregates present in a mixture. Generally, in aprocess of ADC or antibody preparation, one or more polishing steps maybe employed, wherein the polishing step may be selected from affinitychromatography, cation exchange chromatography, anion exchangechromatography, hydrophobic interaction chromatography, mixed modechromatography and hydroxyapatite chromatography.

As used herein, the terms “impurities” and “contaminants” refer toundesirable substances in a mixture of a target molecule, such as cells,cell debris, host cell proteins and other proteins, endotoxins, mediacomponents, lipids, excess reaction materials (such as unreactedlinker-payload intermediates), nucleic acids and viruses.

As used herein, the term “ppm (parts per million)” refers to the amountunits of a contaminant (such as HCP or Protein A) per million units ofthe total mass of a target molecule (such as a target conjugate). Theterm is used to refer to a measure of purity of a target molecule.

The term “ultrafiltration” or “UF” refers to membrane filtrationtechnique which employs controlled pore, semi-permeable membranes toconcentrate or fractionate dissolved molecules. Molecules much largerthan the pores are retained in the feed solution and are concentrated indirect proportion to the volume of liquid that passes through themembrane. The pore size of the ultrafiltration membrane is generallybetween 1-100 nm.

The term “diafiltration” or “DF” refers to a technique that usesultrafiltration membranes to completely remove, replace or lower theconcentration of salts or solvents from solutions containing proteins,peptides, nucleic acids, and other biomolecules. The process selectivelyutilizes permeable (porous) membrane filters to separate the componentsof solutions and suspensions based on their molecular size.Ultrafiltration and diafiltration can be used in combination andreferred to as UF/DF.

Virus inactivation is included in purification process of manybiotherapeutics to ensure safety. Several virus inactivation techniquesare known in the art including, temperature, pH, radiation and exposureto certain chemical agents. Typically, virus inactivation could beperformed by low-pH treatment. For Fc fragment-containing molecules,virus inactivation could be performed, for example, following achromatography process step (e.g., Protein A affinity chromatography orcation exchange chromatography). In such cases, the pool containing thetarget molecule is adjusted to the pH desired for virus inactivation andheld there for a certain length of time (viral inactivationacidification (VIA) step), the combination of pH and time having beenshown to result in virus inactivation. The VIA pool is adjusted to a pHvalue close to neutral (viral inactivation neutralization (VIN) step)for further downstream processing.

Virus filtration (also known as virus-retentive filtration) is a commonstep of the purification process of many biotherapeutics. Virusfiltration could be performed by UF or nanofiltration. Comparing withother dedicated virus clearance unit operations such as low pH or heattreatment, in most cases virus filtration is gentler, thus with lesspotential adverse impacts on product quality. Commercially availablevirus filtration products could be applied, according to the sizes ofthe viruses to be removed.

The term “process step” or “unit operation,” as used interchangeablyherein, refers to the use of one or more methods or devices to achieve acertain result in a purification process.

The term “continuous process,” as used herein, refers to a process forpurifying a target molecule, which includes two or more process steps(or unit operations), such that the output from one process step flowsdirectly into the next process step in the process, without interruptionand/or without the need to collect the entire volume of the output froma process step before performing the next process step. Continuousprocesses, as described herein, also include processes where the inputof the fluid material in any single process step or the output isdiscontinuous or intermittent. Such processes may also be referred to as“semi-continuous” processes.

Ligase Fusion Proteins

In a general aspect, provided is a ligase fusion protein comprising aligase and a Halo tag.

Ligase

The ligase of the present disclosure can be any ligase of interest.Particularly, it can specifically catalyze the conjugation between afirst moiety comprising the recognition motif of the ligase donorsubstrate and a second moiety comprising the recognition motif of theligase acceptor substrate to produce a target conjugate.

In some embodiments, the ligase is a transpeptidase. The transpeptidasecan be naturally occurring or engineered. In some preferred embodiments,the ligase is a sortase, such as a sortase A (SrtA), sortase B (SrtB),sortase C (SrtC), sortase D (SrtD), sortase E (SrtE) or sortase F(SrtF), but not limited to. A “sortase” or “sortase enzyme” hereinrefers to an enzyme having sortase activity to catalyze atranspeptidation reaction, including for example, class A, class B,class C, class D, class E and class F sortases of the sortase enzymesuperfamily (see, e.g., Dramsi, et al., Sorting sortases: a nomenclatureproposal for the various sortases of Gram-positive bacteria, Research inMicrobiology, (2005), 156: 289-297; Bradshaw, et al., Molecular featuresof the sortase enzyme family, FEBS Journal, (2015), 282: 2097-2114;Malik and Kim, A comprehensive in silico analysis of sortasesuperfamily, J Microbiol., (2019), 57(6): 431-443; and EP3647419A1), butnot limited thereto. Such an enzyme may be referred to as a SrtA, SrtB,SrtC, SrtD, SrtE or SrtF, but not limited thereto. A sortase can benaturally occurring or engineered. Naturally occurring sortase enzymescan be found in a variety of gram-positive bacteria, such as any strain,species or subspecies of the genera of Streptococcus (e.g.,Streptococcus pneumoniae and Streptococcus pyogenes), Staphylococcus(e.g., Staphylococcus argenteus and Staphylococcus aureus), Bacillus(e.g., Bacillus anthracis) and Listeria (e.g., Listeria monocytogenes),but are not limited to. An engineered sortase, such as a sortase variantwith substitutions, deletions or insertions of one or more amino acidresidues, can be obtained from its natural counterpart via methods knownin the art, such as protein engineering and chemical synthesis. Alsocontemplated are other variants (such as those with one or more activegroups or labels) of any wild-type sortase known in the art. Theprovision is that the variant has identical or similar function of thewild-type sortase. Those skilled in the art will readily be able toidentify a sortase and assign it to a specific class based on itssequence and other characteristics. However, the definition of sortaseis not limited by any classification method or nomenclature system.

In some particular embodiments, the ligase is a sortase A (SrtA). A SrtAcan be naturally occurring or engineered. Examples of SrtA include thosedescribed in, for example, U.S. Pat. No. 7,238,489 and Malik and Kim,2019, supra, such as those from any strain, subspecies or species of thegenera of Streptococcus (e.g., Streptococcus pneumoniae andStreptococcus pyogenes), Staphylococcus (e.g., Staphylococcus argenteusand Staphylococcus aureus), Streptomyces (e.g., Streptomycescoelicolor), Bacillus (e.g., Bacillus anthracis), Lactobacillus (e.g.,Lactobacillus plantarum) and Listeria (e.g., Listeria monocytogenes),but are not limited to. The amino acid sequences of various SrtA can befound in, for example, U.S. Pat. No. 7,238,489 or public sequencedatabases (such as GenBank and Uniprot), the relevant content of whichis incorporated herein by reference. Exemplary amino acid sequences ofnaturally occurring SrtA useful in the present disclosure can beproteins of Uniprot Accession Numbers: Q2FV99, A0A3S0JRJ4, A0A2T4Q430,A0A507SMZ3, A0A1F2JEX6, A0A364UNR7, A0A1J3ZU75, A0AOM2NSU2, A0A432A5V1,A0A1J4HB57, A0A4Q8MXV4, W1W5Z3, A0A2T4KDK7, A0A2K4DQX6, A0A2T4KHW3,A0A380FYB6, A0A2K4COY9, A0A4Q9WQB8, A0A121AFU6, A0A1Q8DH59, A0A5B2YTH7,A0A533IYI6, Q4L923, A0A1F1M8Z4, A0A2A1KC84 and A0A133Q671, but are notlimited to. Engineered SrtA have been reported in various literatures,for example, WO 2016/014501, the relevant content of which isincorporated herein by reference. For example, an engineered SrtA havingone or more substitutions (such as Pro94Arg, Asp160Asn, Asp165Ala,Lys190Glu, Lys196Thr, Glu105Lys and Glu108G1n) when compared to Q2FV99,or a truncated SrtA with an N-terminal 59 amino acids deletion comparedto Q2FV99 as described in WO 2016/014501 may be considered. The aminoacid sequence of a SrtA variant can have a sequence identity of at leastabout 85%, at least about 90%, at least about 95%, or at least about 99%with any other amino acid sequence described above. Also contemplatedare variants (such as those with one or more active groups or labels) ofany wild-type SrtA known in the art. The provision is that the varianthas identical or similar function of the wild-type SrtA.

In some embodiments, the SrtA comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-26 (WT). In some otherembodiments, the SrtA comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-26 and comprises amino acidsubstitutions at positions 34, 100, 105 and 136. In some embodiments,the amino acid residues at positions 34, 100, 105 and 136 aresubstituted with Ser, Asn, Ala and Thr (i.e.,[Ser34][Asn100][Ala105][Thr136], SNAT), Tyr, Asn, Ala and Thr (i.e.,[Tyr34][Asn100][Ala105][Thr136], YNAT), Trp, Asn, Asp and Thr (i.e.,[Trp34][Asn100][Asp105][Thr136], WNDT), or Val, Asn, Asn and Ser (i.e.,[Val34][Asn100][Asn105][Ser136], VNNS), respectively. In a particularembodiment, the sortase A comprises the amino acid sequence of SEQ IDNO: 27, which is the SNAT counterpart of SEQ ID NO: 1.

In some embodiments, the sortase A comprises an amino acid sequencehaving a sequence identity of at least about 85%, at least about 90%, atleast about 95%, or at least about 99% with an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-26.

In some embodiments, the sortase A comprises an amino acid sequencehaving a sequence identity of at least about 85%, at least about 90%, atleast about 95%, or at least about 99% with an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-26 and comprisesamino acid substitutions of SNAT, YNAT, WNDT or VNNS at positions 34,100, 105 and 136.

In another aspect, provided is a SrtA comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-26 and comprisingamino acid substitutions of SNAT, YNAT, WNDT or VNNS at positions 34,100, 105 and 136 or an amino acid sequence having a sequence identity ofat least about 85%, at least about 90%, at least about 95%, or at leastabout 99% thereto. In another particular aspect, provided is a SrtAcomprising the amino acid sequence of SEQ ID NO: 27 or an amino acidsequence having a sequence identity of at least about 85%, at leastabout 90%, at least about 95%, or at least about 99% thereto.

Halo Tag

A Halo tag is a mutant haloalkane dehalogenase or a variant thereof thatremoves the halogen from a haloalkyl substrate (e.g., an agentcomprising a haloalkyl moiety —(CH₂)₂₋₃₀—X, wherein X is a halogen likeF, Cl, Br, I, particularly Cl or Br) and forms covalent linkage with theremaining moiety of the substrate. Mutant haloalkane dehalogenases havebeen described in, for example, WO 2006/093529 and WO 2008/054821, therelevant content of which is incorporated herein by reference. Mutanthaloalkane dehalogenases useful in the present disclosure may include,but are not limited to, mutants of Xanthobacter dehalogenases (such asXanthobacter autotrophicus dehalogenase (DhIA)) or Rhodococcusdehalogenases (such as Rhodococcus rhodochrous dehalogenase (DhaA)),such as those comprising one or more substitutions at the catalytictriad residues, such as substitution of His272 with Phe/Ala/Gly/Gln/Asnor Asp106 with Cys or other substitutions as described in WO2008/054821. The provision is that the mutant haloalkane dehalogenase isable to form covalent linkage with a haloalkyl substrate.

In some preferred embodiments, the Halo tag comprises the amino acidsequence of SEQ ID NO: 28. In some embodiments, the Halo tag comprisesan amino acid sequence having at least about 85%, at least about 90%, atleast about 95%, or at least about 99% sequence identity with SEQ ID NO:28.

Embodiments Further Comprising Additional Elements and/or Modifications

Optionally, the ligase fusion protein may further comprise one or moreadditional elements, such as an additional polypeptide or a label.Preferably, the ligase fusion protein substantially preserves thedesired properties. Those of skills in the art can select a suitableelement based on the desired function or property of the fusion protein.Methods for introducing such elements are known in the art.

The additional polypeptide can be a protein tag with a desired property.Examples of protein tags may include, but be not limited to, reporterproteins, binding tags and solubility-enhancement tags. Examples ofreporter proteins include, but are not limited to, fluorescent proteins(e.g., green fluorescent protein and its variants), AP (AlkalinePhosphatase) and HRP (Horseradish Peroxidase). A binding tag caneffectively bind to a corresponding binding partner in a covalent ornon-covalent manner Examples of binding tags include, but are notlimited to, polyhistidine tag (i.e., His tag, e.g., His₆ or His₈ tag),Fc tag (the constant region (domain 3 and 4) of immunoglobulinheavy-chain), calmodulin tag, maltose-binding protein (MBP),glutathione-S-transferase (GST), S tag (which interacts withribonuclease S-protein), peptides that bindavidin/streptavidin/neutravidin (e.g., SBP tag, Strep tag and Strep tagII), Halo tag, SNAP tag and CLIP tag (engineered mutants of the DNArepair protein O⁶-alkylguanine-DNA alkyltransferase) and variantsthereof. A solubility-enhancement tag, when expressed as a part of arecombinant protein, can typically enhance the expression level andsolubility of the recombinant protein. Examples ofsolubility-enhancement tags include, but are not limited to, GB1 tag(the B1 domain of Streptococcal protein G), the Z domain ofStaphylococcal protein A, SUMO (Small ubiquitin-related modifier),thioredoxin, GST and MBP. It is appreciated that the property of aprotein tag does not constitute any limitation on the embodiments, and aprotein tag can have one or more properties, for example, a reporterprotein or a binding tag can be a solubility-enhancement tag as well.

The additional polypeptide can also be a short peptide that can serve asa linker, a spacer or an enzyme-cleavable sequence (such as a TEVprotease recognition motif or a thrombin recognition motif). In someembodiments, a linker peptide (such as a polyglycine stretch, (G₄S)_(n),wherein G is glycine, S is serine, and n is an integer of 1-6,preferably n is an integer of 2-5) which is rigid or flexible may beinserted between the ligase and the Halo tag to ensure the properfunction of the fusion protein using methods known in the art. In someembodiments, the linker peptide is (G₄S)₂. In some embodiments, theligase fusion protein comprises the amino acid sequence of SEQ ID NO:29.

The label can be a tracer molecule, such as a fluorophore, aradionuclide, a fluorescent molecule, a fluorescent quantum dot or ananogold particle. The label can also be an affinity label, such asBiotin. Such labels may be used to monitor reactions catalyzed by thefusion protein or to track or immobilize the fusion protein.

The ligase fusion protein may comprise one or more modifications,wherein the ligase, the Halo tag and the additional polypeptide (whenapplicable) are independently modified through, for example,substitution, deletion, addition, insertion of one or more amino acids,or introduction of moieties or active groups at one or more suitableresidues, as long as the desired biological activities or functions ofthe modified fusion protein are substantially similar with that of thecorresponding fusion protein.

Specific Embodiments for Ligases Having an Alkaline pI

In a preferable aspect, provided is a ligase fusion protein having analtered pI comparing to the ligase from which it is derived, wherein theligase has an alkaline pI and the Halo tag has an acidic pI. By fusingto a ligase with an alkaline pI to a Halo tag with an acidic pI, aligase fusion protein with an altered pI is obtained, thereby leading tocertain beneficial effects under certain circumstances. For example, theligase fusion protein may have altered charge characteristics comparingto the ligase under certain conditions (for example, in a specificbuffer system at a given pH or in an in vivo environment), therebyleading to, for example, altered solubility, stability or electrostaticinteraction patterns (i.e., the ability to form electrostaticinteraction with a charged substance) comparing to the ligase.

In some embodiments, the ligase has an isoelectric point (pI) of about7.5 to about 10.0, the Halo tag has a pI of about 4.5 to about 5.0, andthe pI of the ligase fusion protein is about 2.0 to about 4.5 pH unitslower than that of the ligase. In those embodiments comprising one ormore additional elements (such as additional polypeptides or labels asdefined above or a combination thereof) and/or modifications (such asamino acid substitutions, deletions, additions, insertions, or moietiesor active groups), it is preferable that the desired pI differencebetween the ligase fusion protein and the ligase is achieved. In someembodiments, the additional polypeptide (if applicable) may have aspecific pI that helps to achieve the desired pI of the ligase fusionprotein.

In some embodiments, the pI of the ligase fusion protein is about 2.0 toabout 2.5 pH units lower than that of the ligase, such as about 2.0,2.1, 2.2, 2.3, 2.4 or 2.5 pH units lower than that of the ligase. Insome embodiments, the pI of the ligase fusion protein is about 2.6 toabout 3.0 pH units lower than that of the ligase, such as about 2.6,2.7, 2.8, 2.9 or 3.0 pH units lower than that of the ligase. In someembodiments, the pI of the ligase fusion protein is about 3.1 to about3.5 pH units lower than that of the ligase, such as about 3.1, 3.2, 3.3,3.4 or 3.5 pH units lower than that of the ligase. In some embodiments,the pI of the ligase fusion protein is about 3.6 to about 4.0 pH unitslower than that of the ligase, such as about 3.6, 3.7, 3.8, 3.9 or 4.0pH units lower than that of the ligase. In some embodiments, the pI ofthe ligase fusion protein is about 4.1 to about 4.5 pH units lower thanthat of the ligase, such as about 4.1, 4.2, 4.3, 4.4, 4.5 pH units lowerthan that of the ligase.

In some embodiments, the pI of the ligase fusion protein is about 4.5 toabout 6.5, such as about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5. In somepreferred embodiments, the pI of the ligase fusion protein is about 5.0to about 6.0.

In some embodiments, the pI of the ligase is about 7.5 to about 8.5,such as 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5. In someembodiments, the pI of the ligase is about 8.6 to about 9.5, such as8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4 or 9.5. In some embodiments,the pI of the ligase is about 9.6 to about 10.0, such as about 9.6, 9.7,9.8, 9.9 or 10.0.

In some particular embodiments, the pI of the fusion protein is about5.0 to about 6.0, and the pI of the ligase is about 7.6 to about 9.7.

In some embodiments, the ligase is a sortase. The sortase can beselected from SrtA, SrtB, SrtC, SrtD, SrtE and SrtF.

In some preferred embodiments, the SrtA comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-12 (WT). In someother embodiments, the SrtA comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-12 and comprises amino acidsubstitutions at positions 34, 100, 105 and 136. In some embodiments,the amino acid residues at positions 34, 100, 105 and 136 aresubstituted with Ser, Asn, Ala and Thr (SNAT), Tyr, Asn, Ala and Thr(YNAT), Trp, Asn, Asp and Thr (WNDT), or Val, Asn, Asn and Ser (VNNS),respectively. The pIs of these SrtA are listed in Table 1.

TABLE 1 WT SNAT YNAT WNDT VNNS SEQ ID NO: 1 7.673 8.508 8.473 7.6758.508 SEQ ID NO: 2 8.867 9.114 9.066 8.874 9.114 SEQ ID NO: 3 9.4459.458 9.403 9.306 9.458 SEQ ID NO: 4 8.874 9.123 9.075 8.881 9.123 SEQID NO: 5 8.867 9.114 9.066 8.874 9.114 SEQ ID NO: 6 9.114 9.306 9.2559.123 9.306 SEQ ID NO: 7 8.868 9.114 9.066 8.874 9.114 SEQ ID NO: 88.867 9.114 9.066 8.874 9.114 SEQ ID NO: 9 9.132 9.471 9.414 9.319 9.471SEQ ID NO: 10 8.896 9.33 9.275 9.141 9.33 SEQ ID NO: 11 9.414 9.6599.605 9.544 9.659 SEQ ID NO: 12 8.896 9.33 9.275 9.141 9.33

In some embodiments, the sortase A comprises an amino acid sequencehaving a sequence identity of at least about 85%, at least about 90%, atleast about 95%, or at least about 99% with an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-12.

In some embodiments, the sortase A comprises an amino acid sequencehaving a sequence identity of at least about 85%, at least about 90%, atleast about 95%, or at least about 99% with an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-12 and comprisesamino acid substitutions of SNAT, YNAT, WNDT or VNNS at positions 34,100, 105 and 136.

In a particular embodiment, the sortase A comprises the amino acidsequence of SEQ ID NO: 27. It is the SNAT counterpart of SEQ ID NO: 1and has a pI of 8.508.

In some embodiments, a linker peptide (such as a polyglycine stretch,(G₄S)_(n), wherein G is glycine, S is serine, and n is an integer of1-6, preferably n is an integer of 2-5) which is rigid or flexible maybe inserted between the ligase and the Halo tag to ensure the properfunction of the fusion protein using methods known in the art. In someembodiments, the linker peptide is (G₄S)₂. In some embodiments, theligase fusion protein comprises the amino acid sequence of SEQ ID NO:29.

Methods for Obtaining the Ligase Fusion Protein

The ligase, the Halo tag and the additional polypeptide (whenapplicable) can be fused in any manner. In some embodiments, the ligaseis N-terminal to the Halo tag. In some embodiments, the Halo tag isN-terminal to the ligase. In some embodiments, a linker peptide (such asa polyglycine stretch, (G₄S)_(n), wherein G is glycine, S is serine, andn is an integer of 1-6, preferably n is an integer of 2-5) which isrigid or flexible may be inserted between the ligase and the Halo tag toensure the proper function of the fusion protein using methods known inthe art. In some embodiments, the linker peptide is (G₄S)₂. In someembodiments, the ligase fusion protein comprises the amino acid sequenceof SEQ ID NO: 29.

The ligase fusion protein can be obtained using various techniques knownin the art, such as expressed from a nucleic acid obtained byrecombinant DNA techniques, chemical synthesis, enzyme-catalyzedcoupling or chemical coupling methods. In some preferred embodiments,the ligase fusion protein is a recombinant protein encoded by a nucleicacid comprising nucleic acid sequences encoding the ligase and the Halotag. The recombinant protein can be expressed in and purified from asuitable host cell, such as a mammalian cell, a bacterium, a yeast cellor an insect cell, preferably a bacterium, such as E. coli.

Nucleic Acids and Vectors

Also provided is a nucleic acid encoding the ligase fusion proteinaccording to the present disclosure, comprising a first polynucleotideencoding the ligase and a second polynucleotide encoding the Halo tagaccording to the present disclosure, wherein the first and secondpolynucleotides are operably linked to a promoter. In some embodiments,the nucleic acid according to the present disclosure further comprises athird polynucleotide encoding an additional polypeptide operably linkedto the ligase and the Halo tag. Examples of the third polypeptide are asdescribed above.

In some embodiments, the first polynucleotide encodes a sortase A, andthe second polynucleotide encodes a Halo tag. In some embodiments, thefirst polynucleotide encodes a sortase A having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-26, and the secondpolynucleotide encodes a Halo tag having the amino acid sequence of SEQID NO: 28. In a particular embodiment, the first polynucleotide encodesa SrtA having the amino acid sequence of SEQ ID NO: 27 and the secondpolynucleotide encodes a Halo tag having the amino acid sequence of SEQID NO: 28. In another particular embodiment, the nucleic acid encodes aligase fusion protein having the amino acid sequence of SEQ ID NO: 29.It is appreciated by those of skill in the art that one or morenucleotides in the nucleic acid can be optimized without departing fromthe spirit of the present disclosure.

In some embodiments, the nucleic acid according to the presentdisclosure is prepared as recombinant nucleic acids, which may furthercomprise one or more additional polynucleotides, such as regulatoryelements and polynucleotides encoding protein tags. Such regulatoryelements may regulate the expression of the fusion protein according tothe present disclosure, including, but not limited to, enhancers,insulators, internal ribosome entry sites (IRES). The recombinantnucleic acids comprising the nucleic acid according to the presentdisclosure can be prepared using molecular cloning techniques well knownin the art, for example, chemical synthesis, site-directed mutagenesisand polymerase chain reaction (PCR) techniques (see Sambrook, J., E. F.Fritsch, and T. Maniatis (1989), Molecular cloning: a laboratory manual,2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In some embodiments, the nucleic acid according to the presentdisclosure is cloned into a vector, preferably, an expression vectorexpressible in a host cell (e.g., a bacterial, mammalian, yeast orinsect cell). Those skilled in the art are able to select suitableexpression vector based on the nature of the ligase fusion protein andthe host cell to be used. In some embodiments, the vector is a bacterialexpression vector expressible in bacteria, such as E. coli. In someembodiments, the expression vector can further contain one or moreselective marker genes, for example, a neomycin or puromycin resistancegene. After expression, the fusion protein can be purified using methodsknown in the art according to the protein tag used. Depending on thetype of host cells and purification strategies to be used, those ofskills in the art are able to select suitable expression vectors,promoters, regulatory elements and protein tags.

Immobilized Ligase

In another general aspect, provided is an immobilized ligase, comprisingthe ligase fusion protein according to the present disclosureimmobilized to a support.

The support may be in solid form or semi-solid form made of anymaterial. Non-limiting examples of the support may include, but are notlimited to, a resin (e.g., an agarose resin, silicone resin, polymethylmethacrylate resin, epoxy resin or cellulose resin), gel (such as analginate hydrogel), a bead/microsphere/particle (e.g., a polystyrenebead, a magnetic particle), a plate, a well, a tube, a film, a membrane,a matrix and glass (e.g., a glass slide).

In some preferred embodiments, the support is a resin. In some morepreferred embodiments, the support is selected from the group consistingof agarose resin, silicone resin, polymethyl methacrylate resin andcellulose resin. In a particular embodiment, the support is a highlycrosslinked agarose resin.

Methods of enzyme immobilization are known in the art, such asadsorption, covalent or non-covalent binding, entrapment, encapsulation,and cross linking. It is desirable that a maximum enzymatic activity ofthe ligase is preserved after immobilization and a minimum amount offree ligase is present in the conjugate product after the conjugationreaction. Preferably, the support is modified on the surface to compriseone or more functional groups such that the ligase fusion protein can becovalently immobilized on the support.

Preferably, the support comprises one or more chemically activefunctional groups that can form covalent bond with reactive groups (suchas amines, thiols and carboxylates) of the ligase fusion protein or withreactive groups in a haloalkyl substrate, or the support comprises oneor more binding partners of a corresponding binding tag/affinity labelthat is comprised in the ligase fusion protein. Correspondencerelationship between chemically active functional groups and thereactive groups or correspondence relationship between bindingtags/affinity labels and binding partners are well-known in the art.

In some embodiments, the support comprises chemically active functionalgroups that can form covalent bond with reactive groups (such as amines,thiols and carboxylates) on the ligase fusion protein or with reactivegroups in a haloalkyl substrate. In some particular embodiments, thesupport comprises functional groups selected from the group consistingof cyanate ester, isothiocyanates, isocyanates, carbodiimides,N-Hydroxysuccinimide (NHS) esters, amines, carbonates, epoxides,maleimides, haloacetyls, aziridines, ethyl chloroformate and aliphaticaldehydes.

In some embodiments, the support is an epoxy-activated resin, a CNBr(cyanogen bromide)-activated resin or an NHS-activated resin, preferablyan epoxy-activated resin. In some particular embodiments, the support isan epoxy-activated agarose resin, preferably an epoxy-activated highlycrosslinked agarose resin. In some preferred embodiments, beforereacting with the haloalkyl substrate, the epoxy-activated resin ispre-processed to introduce amino group. In some preferred embodiments,the pre-processing of the epoxy-activated resin is performed usingammonia. In some preferred embodiments, the pre-processing of theepoxy-activated resin results in introduction of amino group on theoxirane ring and ring-opening of the oxirane ring gives a hydroxy group.Such hydroxy group are optionally end-capped in subsequent procedures ofthe preparation of the support. In a particular embodiment, thepre-processing of the epoxy-activated resin results in introduction ofamino group on the oxirane ring and ring-opening of the oxirane ringgives a hydroxy group which is optionally esterified usingesterification agent (e.g., acetylation agent like Ac₂O) in subsequentprocedures of the preparation of the support. Such preprocessedepoxy-activated resin is within the scope of “epoxy-activated resin” asdefined above. In some preferred embodiments, the resin is an agaroseresin (such as a highly crosslinked agarose resin) or a polymethylmethacrylate resin.

In some other embodiments, the support comprises one or more bindingpartners of a corresponding binding tag/affinity label that is comprisedin the ligase fusion protein, such as an additional tag or affinitylabel. Correspondence relationship between reactive groups or betweenbinding tags/affinity labels and binding partners are well-known in theart. Examples of binding tags/affinity labels and the correspondingbinding partners may include, but are not limited to, His tag and Ni²⁺,biotin/SPB tag/Strep tag/Strep tag II andstreptavidin/avidin/neutravidin, GST tag and Glutathione, Fc tag andProtein A, calmodulin tag and Ca²⁺, MBP and amylose, S tag andribonuclease S-protein, SNAP tag and benzylguanine (BG) derivatives, andCLIP tag and benzylcytosine (BC) derivatives.

In some preferred embodiments, the support is functionalized to formcovalent interaction with the Halo tag by comprising haloalkyl linkers.The haloalkyl linkers can be introduced to the support by covalentlyconnecting one or more functional groups comprised by the support to oneor more reactive groups in haloalkyl substrates, and the supportobtained thereby is also known as a haloalkyl linker-modified support.The haloalkyl linker-modified support is within the scope of “support”as defined above. Examples of haloalkyl substrates include but are notlimited to those described in e.g., US20060024808A1 and WO2006093529.Haloalkyl substrates and methods for preparing such supports aredescribed in, for example, U.S. Pat. Nos. 7,429,472, 7,888,086 and8,202,700, Japanese Pat. No. 4748685, the relevant content of which isincorporated herein by reference.

The haloalkyl substrate may comprise a haloalkyl moiety comprising aprimary or secondary halo group, preferably a primary halo group. Thehalo group in the haloalkyl moiety is selected from F, Cl, Br and I,preferably selected from Cl and Br. In some embodiments, the haloalkylsubstrate has a structure of the following formula (I):

(F1_(a)-H1_(b))_(r)-Lh-(F2_(b)-H2_(a))s  (I)

wherein,

F1 and F2 are independently a moiety comprising a reactive group whichcan form covalent bond with chemically active functional groupscomprised by the support;

H1 and H2 are independently selected from halo C₂₋₃₀ alkyl;

Lh is a chemical bond or is a C₃₋₂₀₀ alkylene, and wherein one or more(—CH₂—) structures in the alkylene is optionally replaced by —O—, —NH—,—(CO)—, —NH(CO)— and —(CO)NH—;

Lh is optionally substituted with 1, 2 or 3 substituents selected from—O—C₁₋₁₀ alkyl, —NH—C₁₋₁₀ alkyl, —(CO)—C₁₋₁₀ alkyl, —NH(CO)-C₁₋₁₀ alkyland —(CO)NH—C₁₋₁₀ alkyl;

a is 0 or 1, b is 0 or 1, provided that a and b are different;

r is an integer of 1 to 100;

s is an integer of 1 to 100.

In some embodiments, r is an integer of 1 to 10, for example, 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In some embodiments, s is an integer of 1 to 10,for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments,reactive group in F1 or F2 is selected from amino, amines, thiol group,thiols and active esters. In some embodiments, the active ester containsone or more carboxylic acid radicals (such as in carbonic acid monoesterof suitable alcohol or phenol, e.g. electron-deficient phenol like4-nitrophenol; or such as in NHS esters or sulfo-NHS esters) or one ormore sulfonic acid radicals (such as in methane sulfonic acid activeester, e.g., MsO—).

In a particular embodiment, F1 or F2 is

In some embodiments, H1 and H2 are independently selected from haloC₂₋₂₀ alkyl, preferably halo C₂₋₁₀ alkyl, especially halo C₆ alkyl. Insome particular embodiments, the alkyl in H1 or H2 is a linear alkyl. Ina particular embodiment, H1 or H2 is (CH₂)₂₋₃₀—X, preferably(CH₂)₂₋₂₀—X, more preferably (CH₂)₂₋₁₀—X, especially (CH₂)₆—X, wherein Xis a halogen selected from F, Cl, Br and I.

In some preferred embodiments, the support is HaloLink™ resin (Promega).

In some more preferred embodiments, the support is a resin that maycomprise a haloalkyl linker comprising the structure of —(CH₂)₂₋₃₀—X,wherein X is a halogen selected from F, Cl, Br and I. In a particularembodiment, the support is a haloalyl linker-modified resin, preferablyan agarose resin or a polymethyl methacrylate resin, more preferably ahighly crosslinked agarose resin.

In some particular embodiments, a is 1, b is 0, r is 1, s is 1, F1 is

Lh is

H2 is (CH₂)_(2_20)—Cl, and the haloalkyll substrate is a chloroalkylsubstrate having the structure of formula (I-1):

wherein, u is an integer of 1 to 20, v is an integer of 0 to 20, and wis an integer of 1 to 19.

In a particular embodiment, u is 3, v is 2, and w is 5, and thechloroalkyl substrate has the following structure of formula (I-1-1):

In a particular embodiment, the support is a chloroalkyl linker-modifiedsupport and has the structure of formula (II):

wherein u is an integer of 1 to 20, v is an integer of 0 to 20, and w isan integer of 1 to 19;

depicts the support and is a resin, a bead, a membrane, a gel, a matrix,a film, a plate, a well, a tube, a glass slide or a surface, preferablya resin, more preferably an agarose resin, a silicone resin, apolymethyl methacrylate resin or cellulose resin, and most preferably ahighly crosslinked agarose resin. Note that for the sake of clarity,only a single chloroalkyl-linker moiety is depicted attached to thesupport, but it is understood that there would be many suchchloroalkyl-linker moieties attached to the support.

In an embodiment, the chloroalkyl linker-modified support as shown informula (II) is prepared using a resin, a bead, a membrane, a gel, amatrix, a film, a plate, a well, a tube, a glass slide or a surface asdenoted by

with the chloroalkyl substrate of formula (I-1).

In a particular embodiment, the chloroalkyl linker-modified support asshown in formula (II) is prepared from a preprocessed epoxy-activatedresin, which is prepared by introduction of amino group on the oxiranering of the epoxy-activated resin, and ring-opening of the oxirane ringduring the preprocessing gives a hydroxy group which is optionallyesterified using Ac₂O in subsequent procedures of the preparation of thesupport, and the support as shown in formula (II) has the structure offormula (II-1):

wherein,

the substructure

represents the preprocessed epoxy-activated resin, wherein the moiety

represents an oxirane ring which is reacted with amino group andring-opened to give a hydroxy group esterified subsequently to formAcO—, and the moiety

represents the other part of the preprocessed epoxy-activated resin.

In some embodiments, the immobilized ligase has the following structure:

Support ---- Linker ---- HaloTag Ligase

wherein

Support is a solid support, e.g. selected from resin, a bead, amembrane, a gel, a matrix, a film, a plate, a well, a tube, a glassslide or a surface, preferably a resin, more preferable an agaroseresin, a silicone resin, a polymethyl methacrylate resin or celluloseresin, most preferably a highly crosslinked agarose resin;

Linker is a linker moiety, covalently bound to the Support, e.g.,comprising a chain of 10 to 60 carbon atoms, optionally comprising oneor more ether, ester, carbamate, and/or amide bonds; e.g., a linkermoiety of Formula (II-1′) or (II′)

wherein u is an integer of 1-20, v is an integer of 0-20, and w is aninteger of 1 to 19;

HaloTag is a Halo tag (haloalkane dehalogenase polypeptide), covalentlybound to the linker;

Ligase is a ligase polypeptide;

wherein one or more “---- Linker ---- HaloTag ---- Ligase” moieties arebound to the same Support.

In some embodiments, an immobilized ligase comprising the linker moietyof Formula (II-1′) is obtained from the following reactions: 1) reactionof one or more chloroalkyl substrates with the support, forming achloroalkyl linker-modified support; and 2) the subsequent reaction ofthe chloroalkyl linker-modified support with HaloTag (for example, theHalo tag comprised in the ligase fusion protein), obtaining theimmobilized ligase.

Use of the Ligase Fusion Protein and the Immobilized Ligase

Also provided is use of the ligase fusion protein or the immobilizedligase according to the present disclosure in the preparation of aconjugate. The type of the conjugate is not limited. The conjugate canbe obtained by contacting the ligase fusion protein or the immobilizedligase with a first moiety and a second moiety, wherein one of the firstmoiety and second moiety comprises the recognition motif of the ligasedonor substrate, and the other comprises the recognition motif of theligase acceptor substrate.

In some embodiments, the conjugate is a bioconjugate. Examples ofbioconjugates may include, but are not limited to, siRNA conjugates,peptide-hormone conjugates, peptide-peptide conjugates, peptide-drugconjugates, antibody-drug conjugates and multispecific antibodies. Insome embodiments, the conjugate comprises a receptor, an antibody or anantibody fragment. In some embodiments, the conjugate is anantibody-drug conjugate.

In some embodiments, the pI of the conjugate is about 1.0 to about 4.0pH units higher than that of the ligase fusion protein, such as about1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9 or 4.0 pH units higher than that of the ligase fusion protein.In some preferred embodiments, the pI of the conjugate is about 2.0 toabout 4.0 pH units higher than that of the ligase fusion protein.

In some embodiments, the pI of the conjugate is about 5.5 to about 10.5,such as about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some preferred embodiments, thepI of the conjugate is about 7.5 to about 10.0. In some particularembodiments, the pI of the conjugate is about 8.0 to about 9.0.

In some embodiments, the pI of the conjugate is about 5.5 to about 10.5,and the pI of the ligase fusion protein is about 4.5 to about 6.5. Insome particular embodiments, the pI of the conjugate is about 8.0 toabout 9.0, and the pI of the ligase fusion protein is about 5.0 to about6.0.

In some embodiments, the ligase fusion protein and the conjugate areseparable from each other using ion exchange chromatography (IEX). TheIEX can be anion exchange chromatography (AEX), cation exchangechromatography (CEX) or a combination thereof. In some otherembodiments, the ligase fusion protein and the conjugate are separablefrom each other using isoelectric focusing, such as iCIEF or CIEF.

In some particular embodiments, the ligase is a sortase, preferably asortase A, and the conjugate is an antibody-drug conjugate.

The Process According to the Present Disclosure

In another aspect, provided is a process for the preparation of aconjugate comprising a first moiety and a second moiety, comprising thesteps of:

(a) providing System 1 comprising the first moiety and providing System2 comprising the second moiety; and

(b) contacting a ligase unit with System 1 and System 2 in step (a) tocatalyze the conjugation reaction between the first moiety and secondmoiety to obtain the conjugate,

wherein the ligase unit comprises a ligase,

the first moiety and the second moiety each independently comprises abiomolecule, a protein, an antibody, an antibody fragment, a receptor, asignal transduction factor, a cell growth factor, a nucleic acid or anucleic acid analogue, a small molecule compound, a glycan, a PEGmoiety, a radionuclide, a cytokine, an immunomodulator, a tracermolecule, a fluorophore, a fluorescent molecule, a peptide, apolypeptide, or a peptidomimetic; and

one of the first moiety and the second moiety further comprises therecognition motif of the ligase donor substrate, and the other one ofthe first moiety and the second moiety comprises the recognition motifof the ligase acceptor substrate.

In one embodiment, the first moiety and the second moiety are connectedwith each other through the coupling of the recognition motif of theligase donor substrate and the recognition motif of the ligase acceptorsubstrate.

In one embodiment, at least one of the first moiety and the secondmoiety comprises a linker, and preferably the ligase recognition motif(i.e., the recognition motif of the ligase donor substrate, or therecognition motif of the ligase acceptor substrate) comprised by thesaid first moiety or the second moiety is a part of the linker. In oneembodiment, the said first moiety or the second moiety comprises apayload and a linker, and the linker may comprise the ligase recognitionmotif and one or more structural moieties which are connected to thepayload. In other embodiment, the said first moiety or the second moietycomprises a biomolecule and a linker, and the linker may comprise theligase recognition motif and one or more structural moieties which areconnected to the biomolecule. In yet another embodiment, the biomoleculeand/or the payload are independently modified to comprise one or moreadditional moieties, such as active groups, spacers and labels.

The terms “first moiety” and “second moiety” of the conjugate are usedherein to refer to various moieties of the conjugate. For example, for abioconjugate, the first moiety can be the biomolecule moiety of theconjugate, and the second moiety can be another functional moiety or therest part of the conjugate. It should be understood that the phase“first” and “second” are only used to designate different moieties forthe purpose of clarity but do not constitute any limitation.

The terms “System 1” and “System 2” are only used to designate differentportions containing the moiety to be conjugated but do not constituteany limitation. System 1 and System 2 can each independently be in anyform, such as aqueous form, solid form or semi-solid form. Preferably,at least one of System 1 and System 2 is aqueous form, such as (aqueous)solution or a fluid. System 1 and System 2 can each independently beselected from a culture (such as a tissue culture, a mammalian cellculture, a yeast cell culture, a bacterial cell culture and abacteriophage culture), a harvested cell culture fluid, a solutioncontaining an antibody, a solution containing a linker-payloadintermediate, etc. “System 1” and “System 2” can be identical ordifferent, preferably they are different.

Ligase Unit

The ligase unit may comprise any ligase without limitation.Particularly, it can recognize the recognition motifs on the twomoieties and catalyze the conjugation between the two moieties. In someembodiments, the ligase is a transpeptidase. In some embodiments, theligase is a sortase. The sortase can be selected from the groupconsisting of SrtA, SrtB, SrtC, SrtD, SrtE, SrtF and a combinationthereof. In some embodiments, the ligase is a SrtA as described above.In some embodiments, the ligase is further modified by comprising one ormore additional elements, such as a protein tag or a label as describedabove, or by comprising one or more amino acid substitutions, deletionsor insertions.

The ligase can be a free ligase or immobilized to a support. Preferably,the ligase is immobilized to a support, such that higher operationalstability and reusability, lower enzyme contamination, less areaoccupancy, and continuous production can be achieved. The support may bein solid form or semi-solid form made of any material. Non-limitingexamples of the support may include, but are not limited to, a resin(e.g., an agarose resin, silicone resin, polymethyl methacrylate resin,epoxy resin or cellulose resin), gel (such as an alginate hydrogel), abead/microsphere/particle (e.g., a polystyrene bead, a magneticparticle), a plate, a well, a tube, a film, a membrane, a matrix andglass (e.g., a glass slide).

Methods for enzyme immobilization are known in the art, such asadsorption, covalent or non-covalent binding, entrapment, encapsulation,and cross linking. It is desirable that a maximum enzymatic activity ofthe ligase is preserved after immobilization and a minimum amount offree ligase is present in the conjugate product after the conjugationreaction. Selection of the support and immobilization method issubjected to the discretion of those of skills in the art.

More preferably, the ligase is covalently immobilized to a support toreduce the amount of free ligase that falls off from the support.Methods for non-specific covalent immobilization of proteins are knownin the art. In some embodiments, the support comprises chemically activefunctional groups that can form covalent bond with reactive groups (suchas amines, thiols and carboxylates) on the ligase. Such functionalgroups can be selected from the group consisting of isothiocyanates,isocyanates, carbodiimides, N-Hydroxysuccinimide (NHS) esters,carbonates, epoxides, maleimides, haloacetyls, aziridines, ethylchloroformate and aliphatic aldehydes.

Most preferably, the ligase is covalently immobilized to a supportthrough a self-labeling protein tag such that a maximum enzymaticactivity is preserved. A self-labeling protein tag is able to formcovalent interaction with its substrate. Such protein tags may include,but are not limited to, SNAP tag, CLIP tag, Halo tag and variantsthereof. Accordingly, the support may comprise the correspondingsubstrate of the protein tag. The correspondence relationship of theprotein tags and their substrates are well known in the art.

In some particular embodiments, the ligase unit comprises the ligasefusion protein according to the present disclosure. In some particularembodiments, the ligase unit comprises the immobilized ligase accordingto the present disclosure.

Conjugate

The process can be used to prepare various kinds of conjugates. In someembodiments, the conjugate is a bioconjugate as described above.

In some embodiments, the conjugate has the structure of formula (III),the first moiety comprises T, and the second moiety comprises alinker-payload intermediate of formula (IV),

wherein

T comprises a biomolecule, which is optionally modified to have one ofthe recognition motif of the ligase donor substrate and the recognitionmotif of the ligase acceptor substrate;

L comprises a linker, which comprises the other of the recognition motifof the ligase donor substrate and the recognition motif of the ligaseacceptor substrate;

P comprises a payload;

z is an integer of 1-20;

t is an integer of 1-20.

t denotes the number of payloads coupled with a single linker to formthe linker-payload intermediate of formula (IV). z denotes the number offormula (IV) compounds coupled with a single T to form the compound offormula (III).

In one embodiment, z is selected from the following values: an integerof 1 to 10, 1 to 8, 1 to 6 or 1 to 4. In another embodiment, z is 1 or2. In a very special embodiment, z is 2.

Biomolecule

In the present disclosure, the biomolecule may be selected from thegroup consisting of proteins, peptides, antibodies, antibody fragments,receptors, signal transduction factors, cell growth factors and nucleicacids and analogues. In one embodiment, T optionally comprises one ofthe recognition motif of the ligase donor substrate and the recognitionmotif of the ligase acceptor substrate, or is optionally modified tohave one of such motifs.

In one embodiment, T is a molecule comprising a receptor, an antibody oran antibody fragment, which is optionally modified to have one of therecognition motif of the ligase donor substrate and the recognitionmotif of the ligase acceptor substrate. In another embodiment, T is areceptor, an antibody or an antibody fragment, which is optionallymodified to have one of the recognition motif of the ligase donorsubstrate and the recognition motif of the ligase acceptor substrate. Ina preferable embodiment, T is a molecule comprising an Fc fragment andan antigen-binding fragment of antibody, which is optionally modified tohave one of the recognition motif of the ligase donor substrate and therecognition motif of the ligase acceptor substrate. In anotherembodiment, T is a soluble receptor, which is optionally modified tohave one of the recognition motif of the ligase donor substrate and therecognition motif of the ligase acceptor substrate.

In some embodiments, T is a targeting molecule, which is optionallymodified to have one of the recognition motif of the ligase donorsubstrate and the recognition motif of the ligase acceptor substrate.Targets recognized by the targeting molecules (such as antibodies orantigen-binding fragments thereof) include but are not limited to CD19,CD22, CD25, CD30/TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74,CD79b, CD117/KIT, CD123, CD138, CD142, CD174, CD227/MUC1, CD352,CLDN18.2, DLL3, ErbB2/HER2, CN33, GPNMB, ENPP3, Nectin-4, EGFRvIII,SLC44A4/AGS-5, mesothelin, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4,SLITRK6, HGFR/cMet, SLAMF7/CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1,MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, FGFR2,FGFR3, CA6, CanAg, Integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3,C4.4A, FLT3.

In one embodiment, the targeting molecule is an anti-human HER2 antibodyor antigen-binding fragment thereof, which is optionally modified tohave one of the recognition motif of the ligase donor substrate and therecognition motif of the ligase acceptor substrate. Examples ofanti-human HER2 antibodies include but are not limited to Pertuzumab andTrastuzumab.

In one embodiment, the targeting molecule is one or more selected fromanti-human TROP2 antibodies or antigen-binding fragment thereof, whichis optionally modified to have one of the recognition motif of theligase donor substrate and the recognition motif of the ligase acceptorsubstrate. In a particular embodiment, the anti-human TROP2 antibody isone or more selected from engineered anti-TROP2 antibodies based on hrS7(US20140120035). In another particular embodiment, the anti-human TROP2antibody is one or more selected from engineered anti-TROP2 antibodiesbased on MAAA1181a (US20160297890).

In a preferred embodiment, the anti-human HER2 or TROP2 antibody is arecombinant antibody selected from the group consisting of monoclonalantibody, chimeric antibody, humanized antibody, antibody fragment, andantibody mimic. In one embodiment, the antibody mimic is selected fromthe group consisting of scFv, minibody, diabody, nanobody. For thecoupling with the compound of formula (IV), the targeting molecule ofthe present disclosure may comprise a modified moiety to connect with D1or D2 in the compound of formula (V), namely the moiety in the linkerwhich comprises a recognition motif of the ligase acceptor or donorsubstrate, cf. below. The introduction position of such modified moietyis not limited, for example, when the targeting molecule is an antibody,its introduction position can be, but not limited to, located at theC-terminal or the N-terminal of the heavy chain or light chain of theantibody.

In an alternative embodiment, a modified moiety for the coupling with D1or D2 in the compound of formula (V) can be introduced at a non-terminalposition of the heavy chain or light chain of the antibody using, forexample, chemical modification methods.

In one embodiment, the targeting molecule of the present disclosure isan antibody or antigen-binding fragment thereof, which may compriseterminal modification. A terminal modification refers to a modificationat the C-terminal or N-terminal of the heavy chain or light chain of theantibody, which for example comprises a ligase recognition motif. Inanother embodiment, the terminal modification may further comprisespacer Sp2 comprising 2-100 amino acids, wherein the antibody, Sp2 andthe ligase recognition motif are sequentially linked. In a preferredembodiment, Sp2 is a spacer sequence containing 2-20 amino acids. In aparticular embodiment, Sp2 is a spacer sequence selected from the groupconsisting of GA, GGGS and GGGGSGGGGS, especially GA.

In a preferred embodiment, the light chain of the antibody orantigen-binding fragment thereof includes 3 types: wild-type (LC); theC-terminus modified light chain (LCCT), which is modified by directintroduction of an ligase recognition motif LPXTG and C-terminusmodified light chain (LCCT_(L)), which is modified by introduction ofshort peptide spacers plus the ligase donor substrate recognition motifLPXTG. The heavy chain of the antibody or antigen-binding fragmentthereof includes 3 types: wild-type (HC); the C-terminus modified heavychain (HCCT), which is modified by direct introduction of an ligaserecognition motif LPXTG; and C-terminus modified heavy chain (HCCT_(L)),which is modified by introduction of short peptide spacers plus theligase donor substrate recognition motif LPXTG. X can be any natural ornon-natural single amino acid. When z in the compound of formula (IV) is1 or 2, the combination of the above heavy and light chains can form 8preferred antibody molecules, see the amino acid sequence table.

In a preferred embodiment, the light chain of the antibody orantigen-binding fragment thereof includes 3 types: wild-type (LC); theN-terminus modified light chain (LCNT), which is modified by directintroduction of an ligase recognition motif GGG; and N-terminus modifiedlight chain (LCNT_(L)), which is modified by introduction of shortpeptide spacers plus the ligase acceptor substrate recognition motifGGG. The heavy chain of the antibody or antigen-binding fragment thereofincludes 3 types: wild-type (HC); the N-terminus modified heavy chain(HCNT), which is modified by direct introduction of an ligaserecognition motif GGG; and N-terminus modified heavy chain (HCNT_(L)),which is modified by introduction of short peptide spacers plus theligase acceptor substrate recognition motif GGG.

The conjugates of the present disclosure can further comprise a payload.The payload is as described in the present disclosure.

Linker

In one embodiment, the linker, namely L in formula (III) and formula(V), is a compound of formula (V):

(A1_(p)-D1_(q)-Y)_(t)-Lk-(W-A2_(q)-D2_(p))_(t)  (V)

wherein,

D1 and D2 are independently a moiety comprising a recognition motif ofthe ligase acceptor or donor substrate;

A1 and A2 independently represents a bond connecting to the payload, ora moiety comprising a reactive group which can be coupled with apayload;

Lk is a chemical bond, L₁-L₂-L₃, or L₁-L₂-L₃-L₄, or L₄-L₁-L₂-L₃, or L₄;

L₁ and L₃ are each independently selected from the group consisting of:

—CH₂—, —NH—, —(CO)—, —NH(CO)—, —(CO)NH—; and combination of a C₁₋₄alkylene with one of the following groups: —CH₂—, —NH—, —(CO)—,—NH(CO)—, —(CO)NH—;

L₂ is absent or is a C₇₋₃₄ alkylene, and wherein one or more (—CH₂—)structures in the alkylene is optionally replaced by —O—;

L₁, L₂ and L₃ are each optionally and independently substituted with 1,2 or 3 substituents selected from —OR₁ and —NR₁R₂;

R₁ and R₂ are each independently selected from the group consisting ofhydrogen, —C₁₋₆ alkyl, —(CO)—C₁₋₆ alkyl and —S(═O)₂—C₁₋₆ alkyl;

L₄ is a peptide sequence (amide bond is formed by the condensationreaction of α-amino and carboxyl groups), wherein it contains anoptionally derivatized Lys (Lysine) (number 1-100), or contains anoptionally derivatized Cys (Cysteine) (number 1-100);

Y and W are each independently absent or selected from the groupconsisting of a cleavable sequence, spacer Sp1, and the combinationthereof;

the cleavable sequence comprises an amino acid sequence which can becleaved by enzyme, and the cleavable sequence comprises 1-10 aminoacids;

Sp1 is selected from the group consisting of a spacer sequencecontaining 1-20 amino acids, PAB, and the combination thereof;

-   -   p is 0 or 1, q is 0 or 1, provided that p and q are different,

t is as defined in formula (III).

In one embodiment, the linker of formula (V) is connected to the payloadthrough A1 or A2, and is connected to the biomolecule T through thecoupling of D1 or D2 with the ligase acceptor substrate recognitionmotif comprised by the biomolecule T. Optionally, the ligase recognitionmotif in the biomolecule T exits in the form of a modified moiety, whichis introduced to the biomolecule through, for example, recombinantmethods or chemical modification methods.

In one embodiment, formula (V) is comprised by the first moiety inSystem 1, or comprised by the second moiety in System 2.

In one embodiment, L₁, L₂ and L₃ are independently substituted with 1,2, or 3 substituents selected from —OR₁ and —NR₁R₂. Substitutions occur,for example, on (—CH₃), (—CH₂—) or

structure, especially on (—CH₂—).

In one embodiment, L₂ is a C₇₋₃₄ alkylene, wherein the alkylene is astraight or a branched alkylene group, and optionally one or more of the(—CH₂—) structures in the alkylene can be replaced by —O—, and thealkylene is optionally substituted with 1, 2 or 3 substituents selectedfrom —OR₁ and —NR₁R₂. In yet another embodiment, L₂ is selected fromgroups optionally substituted with 1, 2 or 3 substituents selected from—OR₁ and —NR₁R₂, wherein the said groups are as follows: methylene,ethylene, propylene, butylene, pentylene, hexylene, 1-methylethylene,2-methylethylene, 2-methylpropylene and 2-ethylpropylene.

In another embodiment, L₂ is —(C₂H₄—I)_(i)—C₁₋₄ alkylene; i is aninteger of 2 to 10. “—(C₂H₄—O)_(i)—” represents a structure formed bypolymerization of PEG units, wherein i indicates the number of PEGunits. In another embodiment, L₂ is —(C₂H₄—O)_(i)—C₁₋₂ alkylene. In aparticular embodiment, L₂ is —(C₂H₄—O)_(i)—C₂H₄—. In another embodiment,L₂ is C₁₋₄ alkylene-(O—C₂H₄)_(i). In another embodiment, L₂ is C₁₋₂alkylene-(O—C₂H₄)_(i). In a particular embodiment, L₂ is—C₂H₄—(O—C₂H₄)_(i)—. In one embodiment, i is selected from the followingvalues: 2-10, 2-8, 2-6, 2-4 or 4-6. In a particular embodiment, i is 4.

In another embodiment of L₄, based on the desired number of couplings,the ε-amino of lysine can either be used to introduce a maleimidefunctional group in A1 or A2 moieties by a suitable bifunctionalcrosslinking agent, or be used to form an amido bond with the α-carboxylgroup of another lysine to form a branched chain, and then the α- andε-aminos of the lysine in the branched chain can be used to introducemaleimide groups by a suitable bifunctional crosslinker. And so on, byincreasing the number of the lysine in the main chain and/or branchedside chain, the number of A1 or A2 moieties introduced by such a moietyL₄ can achieve 1-1000.

In another embodiment of L₄, based on the desired number of couplings,the mercapto group of each cysteine can be used to react with amaleimide functional group in A1 or A2. A1 or A2 can thus be connectedto Lk. A1 and A2 each further comprises a reactive group which can becoupled with a payload. By increasing the number of the cysteine in L₄,for example in the main chain and/or branched side chain of L₄, thenumber of A1 or A2 moieties introduced by such a moiety L₄ can achieve1-1000.

In an embodiment, L₄ is optionally derivatized lysine.

In a preferred embodiment, the derivatization of lysine is selected fromthe group consisting of: 1) amidation of the carboxyl group, theresulting amide NH₂ being optionally substituted with a C₁₋₆ alkylgroup; 2) linkage of the carboxyl group and/or the amino group to anamino acid fragment comprising 1-10 amino acids or a nucleotide fragmentcomprising 1-10 nucleotides, wherein the amino acid fragment ispreferably Gly.

In one embodiment, Y and W are each independently absent or selectedfrom the group consisting of a cleavable sequence, spacer Sp1, and thecombination thereof. In a particular embodiment, Y is absent. In anotherparticular embodiment, W is absent. In yet another particularembodiment, Y and W are both absent. In one embodiment, the cleavablesequence comprises an amino acid sequence that can be recognized asenzyme substrate and can be cleaved by the enzyme. In a particularembodiment, the cleavable sequence can be enzymatically cleaved in thelysosomal of the cell. In another particular embodiment, the cleavablesequence can be cleaved by protease, in particular by cathepsins. In yetanother particular embodiment, the cleavable sequence can be cleaved byglutaminase. In one embodiment, the cleavable sequence is selected fromthe group consisting of a cathepsin restriction site, a glutaminaserestriction site, and combinations thereof. In one embodiment, thecleavable sequence is selected from Phe-Lys, Val-Cit, Val-Lys,Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu and the combination thereof.

In one embodiment, Y and W are each independently absent or selectedfrom spacer Sp1. In another embodiment, Sp1 is a spacer sequencecomprising 1-10, preferably 1-6, more preferably 1-4 amino acids. In aparticular embodiment, Sp1 is Leu. In another particular embodiment, Sp1is Gln. In one embodiment, Sp1 is PAB. In yet another embodiment, Y andW are each independently selected from the group consisting ofPhe-Lys-PAB, Val-Cit-PAB, and Val-Lys-PAB.

In one embodiment, the amino acids comprised by Y and/or W may benatural or unnatural. In a particular embodiment, Y is absent, or isamino acid fragment 1 Amino acid fragment 1 comprises 1-30 natural orunnatural amino acids, which are each independently the same ordifferent. And amino acid fragment 1 is selected from the groupconsisting of: a cleavable sequence comprising 1-10 amino acids, aspacer sequence comprising 1-20 amino acids, and the combinationthereof. In another particular embodiment, W is absent, or is amino acidfragment 2. Amino acid fragment 2 comprises 1-30 natural or unnaturalamino acids, which are each independently the same or different. Andamino acid fragment 2 is selected from the group consisting of: acleavable sequence comprising 1-10 amino acids, a spacer sequencecomprising 1-20 amino acids, and the combination thereof.

In one embodiment, p=0, q=1, the structure of the compound of formula(V) is as shown in the following formula (V-1):

D1-Y-Lk-(W-A2)_(t)  (V-1)

wherein, A2, D1, Y, Lk, and W are as defined in formula (V),respectively.

In another embodiment, p=1, q=0, the structure of the compound offormula (III) is as shown in the following formula (V-2):

(A1-Y)_(t)Lk-W-D2  (V-2)

wherein, A1, D2, Y, Lk and W are as defined in formula (V),respectively.

In an embodiment, suitable linker can be selected from those in any oneof FIGS. 13 to 16 in WO2014177042A. In yet another embodiment, suitablelinker can be selected from those in any one of FIGS. 7 to 10 inWO2015165413A.

In an embodiment, suitable linker can be selected from those in any oneof FIGS. 1 to 12 in WO2014177042A. In yet another embodiment, suitablelinker can be selected from those in any one of FIGS. 3 to 6 inWO2015165413A.

Moiety Comprising Recognition Motif of the Ligase Acceptor or DonorSubstrate

In one embodiment, the ligase is a transpeptidase. In one embodiment,the ligase is selected from the group consisting of a naturaltranspeptidase, an unnatural transpeptidase, variants thereof, and thecombination thereof. Unnatural transpeptidase enzymes can be, but arenot limited to, those obtained by engineering of natural transpeptidase.

In a preferred embodiment, the ligase is selected from the groupconsisting of a natural Sortase, an unnatural Sortase, and thecombination thereof. The species of natural Sortase include SrtA, Srt B,SrtC, SrtD, SrtE, SrtF, etc. (see, e.g., US20110321183A1 andEP3647419A1). The type of ligase corresponds to the ligase recognitionmotif and is thereby used to achieve specific coupling between differentmolecules or structural fragments.

In one embodiment, the recognition motif of the ligase acceptorsubstrate is selected from the group consisting of oligomeric glycine,oligomeric alanine, and a mixture of oligomeric glycine/alanine having adegree of polymerization of 3-10. In a particular embodiment, therecognition motif of the ligase acceptor substrate is G_(n), wherein Gis glycine (Gly), and n is an integer of 3 to 10.

In some embodiments, the ligase is a SrtA, and the donor recognitionmotif can be LPXTG, wherein X is any natural or unnatural amino acid. Insome embodiments, the ligase is a SrtB, and the donor recognition motifcan be NPXTG, wherein X is any natural or unnatural amino acid. In someembodiments, the ligase is a SrtC, and the donor recognition motif canbe LPXTG, wherein X is any natural or unnatural amino acid. In someother embodiments, the ligase is a SrtD, and the donor recognition motifcan be LPXTA, wherein X is any natural or unnatural amino acid. In yetsome other embodiments, the ligase is a SrtE, and the donor recognitionmotif can be LAXTG, wherein X is any natural or unnatural amino acid. Insome other embodiments, the ligase is a SrtF, and the donor recognitionmotif can be LPXTG, wherein X is selected from the group consisting ofA, R, N, D, Q, I, L and K.

In another particular embodiment, the ligase is a SrtA fromStaphylococcus aureus. Accordingly, the ligase recognition motif may bethe typical recognition motif LPXTG of the enzyme. In yet anotherparticular embodiment, the recognition motif of the ligase donorsubstrate is LPXTGJ, and the recognition motif of the ligase acceptorsubstrate is G_(n), wherein X can be any single amino acid that isnatural or unnatural; J is absent, or is an amino acid fragmentcomprising 1-10 amino acids, optionally labeled. In one embodiment, J isabsent. In yet another embodiment, J is an amino acid fragmentcomprising 1-10 amino acids, wherein each amino acid is independentlyany natural or unnatural amino acid. In another embodiment, J is G_(m),wherein m is an integer of 1 to 10. In yet another particularembodiment, the recognition motif of the ligase donor substrate isLPETG. In another particular embodiment, the recognition motif of theligase donor substrate is LPETGG. In one embodiment, the ligase is SrtBfrom Staphylococcus aureus and the corresponding donor substraterecognition motif can be NPQTN. In another embodiment, the ligase isSrtB from Bacillus anthracis and the corresponding donor substraterecognition motif can be NPKTG. In yet another embodiment, the ligase isSrtA from Streptococcus pyogenes and the corresponding donor substraterecognition motif can be LPXTGJ, wherein J is as defined above. Inanother embodiment, the ligase is SrtE from Streptomyces coelicolor, andthe corresponding donor substrate recognition motif can be LAXTG. In yetanother embodiment, the ligase is SrtA from Lactobacillus plantarum andthe corresponding donor substrate recognition motif can be LPQTSEQ. Theligase recognition motif can also be other totally new recognitionsequence for transpeptidase optimized by manual screening.

When coupling LPXTGJ with G_(n), the upstream peptide bond of theglycine in the LPXTGJ sequence is cleaved by Sortase A, and theresulting intermediate is linked to the free N-terminal of G_(n) togenerate a new peptide bond. The resulting amino acid sequence isLPXTG_(n). The sequences G_(n) and LPXTGJ are as defined above.

In some particular embodiments, the ligase is a SrtA from Staphylococcusaureus, the donor recognition motif is LPETGG, the acceptor recognitionmotif is GGG.

Moiety Comprising Reactive Group

In one embodiment, A1 and A2 in formula (V) are each independentlyselected from the group consisting of amino compound, maleimide andderivative thereof, thiol compound, pyridyldithiol compound, haloaceticacid (haloacetylic acid), isocyanate. In another embodiment, thereactive groups in A1 and A2 are each independently selected from thegroup consisting of amino group, maleimide group, thiol group,pyridyldithio group, haloacetyl group, and isocyanate group.

In an embodiment, according to the structure of the reactive grouptherein, A1 and A2 can each independently covalently couple with aMichael acceptor (the acceptor molecule of Michael addition) through adisulfide bond, a thioether bond, a thioester bond, or a urethane bond.In a particular embodiment, A1 and A2 are each independently selectedfrom optionally derivatized cysteines.

In another particular embodiment, A1 and A2 are each independentlyselected from optionally derivatized cysteines. In a preferredembodiment, the derivatization of cysteine is selected from the groupconsisting of: 1) amidation of the carboxyl group, the resulting amideNH₂ being optionally substituted with a C₁₋₆ alkyl group; 2) acylationof the amino group; and 3) linkage of the carboxyl group and/or theamino group to an amino acid fragment comprising 1-10 amino acids or anucleotide fragment comprising 1-10 nucleotides, wherein the amino acidfragment is preferably Gly. In a particular embodiment, thederivatization of cysteine refers to amidation or linkage to glycine forthe carboxyl group of cysteine.

In one embodiment, A2 is

wherein x is selected from the group consisting of hydrogen, OH, NH₂, anamino acid fragment comprising 1-10 amino acids, and a nucleotidefragment comprising 1-10 nucleotides. In one embodiment, A1 is

wherein x is selected from the group consisting of hydrogen, an aminoacid fragment comprising 1-10 amino acids, and a nucleotide fragmentcomprising 1-10 nucleotides. In one embodiment, acylation of the aminogroup refers to the substitution with a C₁-6 alkylcarbonyl group for theamino group of cysteine.

In some embodiments of the linking unit of formula (V-1), wherein t is1, D1 is G_(n), G is glycine, A2 is

and the structure of the compound of formula (V-1) is as shown in thefollowing formula (V-1-1):

wherein n is an integer of 3 to 10;

x is selected from the group consisting of hydrogen, OH, NH₂, an aminoacid fragment comprising 1-10 amino acids, a nucleotide fragmentcomprising 1-10 nucleotides;

Lk is L₁-L₂-L₃;

L₁, L₂, L₃, t, Y and W are as defined in formula (V), respectively.

In a preferred embodiment, in formula (V-1-1), x is selected from OH,NH₂ and Gly.

In a particular embodiment, in formula (V-1-1), both Y and W are absent,Lk is L₁-L₂-L₃, L₁ is —NH—, L₃ is —(CO)—, L₂ is —(C₂H₄—O)_(i)—C₂H₄—,i=4, and the structure of the compound of formula (V-1-1) is as shown inthe following formula (V-1-1-1):

In a particular embodiment, in formula (V-1-1), W is absent, Y is L, Lis leucine (Leu), Lk is L₁-L₂-L₃, L₁ is —NH—, L₃ is —(CO)—, L₂ is—(C₂H₄—O)_(i)—C₂H₄—, i=4, and the structure of the linking unit is asfollows (V-1-1-2):

In yet a particular embodiment, in formula (V-1-1), W is absent, Y is Q,Q is glutamine (Gln), Lk is L₁-L₂-L₃, L₁ is —NH—, L₃ is —(CO)—, L₂ is—(C₂H₄—O)_(i)—C₂H₄—, i=4, and the structure of the linking unit is asfollows (V-1-1-3):

In a particular embodiment, in formula (V-1-1), both Y and W are absent,Lk is L₁-L₂-L₃, L₁ is —NH—, L₃ is —(CO)—, L₂ is —C₅H₁₀—, and thestructure of the linking unit is as follows (V-1-1-4):

In yet a particular embodiment, in formula (V-1-1), both Y and W areabsent, Lk is L₁-L₂-L₃, L₁ is —NH—, L₃ is —(CO)—, L₂ is —C₅H₁₀— groupsubstituted with one —NR₁R₂ group, R₁ is hydrogen, R₂ is —(CO)CH₃, andthe structure of the linking unit is as follows (V-1-1-5):

In some embodiments of the linking unit of formula (V-2), when t is 1,D2 is LPXTG and A1 is

the structure of the compound of formula (V-2) is as shown in thefollowing formula (V-2-1):

wherein x is selected from hydrogen, an amino acid fragment comprising1-10 amino acids, a nucleotide fragment comprising 1-10 nucleotides;

Lk is L₁-L₂-L₃;

L₁, L₂, L₃, Y and W are as defined in formula (V), respectively.

In one embodiment, x is hydrogen.

In one embodiment, A1 and A2 are each independently a maleimidefunctional group. The maleimide functional group is introduced into themolecule of formula (V) by a suitable bifunctional cross-linking agent.

In a preferred embodiment, the bifunctional cross-linking agent forintroducing maleimide functional group include, but are not limited to,N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),the “long chain” SMCC analogue N-[alpha-maleimidoacetoxy] succinimideester (AMAS), N-gamma-Maleimidobutyryl-oxysuccinimide ester (GMB S),3-MaleiMidobenzoic acid N-hydroxysucciniMide ester (MB S),6-maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS),N-succinimidyl 4-(4-maleimidophenyl) butyrate (SMPB), Succinimidyl6-[(beta-maleimidopropionamido) hexanoate (SMPH), Succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),N-succinimidyl 11-(maleimido) undecanoate (KMUS), and a bifunctionalcrosslinking agent comprising N-hydroxy succinimide-(polyethyleneglycolalcohol)_(n) (SM (PEG)_(n)), wherein n indicated there are 2, 4, 6, 8,12 or 24 polyethylene glycol (PEG) units. The exemplary maleimidefunctional groups introduced to A1 or A2 immediately after reacting withbifunctional cross-linking agent may be those listed in the followingtable:

Bifunctional Exemplary maleimide cross-linking agent functional groupintroduced to A1 or A2 SMCC

AMAS

GMBS

MBS

EMCS

SMPB

SMPH

LC-SMCC

KMUS

a bifunctional crosslinking agent comprising SM (PEG)_(n)

In one embodiment, A1 and A2 are each independently selected from mc andmcc.

In some embodiments of the linker of formula (V-1), wherein t is 1, D1is G_(n), G is glycine, A2 is mcc, W is absent, Lk is L₄, L₄ is anoptionally derivatized lysine, and the structure of the compound offormula (V-1) is as shown in the following formula (V-1-2):

wherein n is an integer of 3 to 10;

x is selected from the group consisting of hydrogen, OH, NH₂, an aminoacid fragment comprising 1-10 amino acids, a nucleotide fragmentcomprising 1-10 nucleotides;

Y is as defined in formula (V).

In a particular embodiment, in formula (V-1-2), Y is absent, n=3, x isOH, and the structure of the linker is as follows (linker LU104):

Payload

In the present disclosure, the payload may be selected from the groupconsisting of hydrogen, small molecule compounds (e.g., inhibitors andtoxins (such as cytotoxins)), glycans, PEG moieties, radionuclides,cytokines, immunomodulators, nucleic acids and analogues (e.g.,interfering RNAs), tracer molecules (e.g., fluorophores and fluorescentmolecules), polypeptides (e.g., protein tags, bioactive peptides,protein toxins and enzymes), peptidomimetics, antibodies and antibodyfragments.

In some embodiments, the payload is selected from the group consistingof small molecule compounds, immunomodulators, nucleic acids andanalogues, tracer molecules, radionuclides, peptidomimetics, glycans,and PEG moieties.

In some embodiments, the payload is selected from the group consistingof bioactive peptides, cytokines, antibodies, antibody fragments andprotein receptors.

In one embodiment, the payload is selected from the group consisting ofsmall molecule compounds, nucleic acid molecules, and tracer molecules.In a preferred embodiment, the payload is selected from small moleculecompounds. In a more preferred embodiment, the payload is selected fromthe group consisting of cytotoxin and fragments thereof. In anembodiment, the payload is one or more radionuclides. In anotherembodiment, the payload is one or more cytokines. In an embodiment, thepayload is one or more immunomodulators.

In one embodiment, the cytotoxin is selected from the group consistingof drugs that target microtubule cytoskeleton. In a preferredembodiment, the cytotoxin is selected from the group consisting oftaxanes, maytansinoids, auristatins, epothilones, combretastatin A-4phosphate, combretastatin A-4 and derivatives thereof,indol-sulfonamides, vinblastines such as vinblastine, vincristine,vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine,dolastatin 10 and analogues, halichondrin B and eribulin,indole-3-oxoacetamide, podophyllotoxins,7-diethylamino-3-(2′-benzoxazolyl)-coumarin (DBC), discodermolide,laulimalide. In another embodiment, the cytotoxin is selected from thegroup consisting of DNA topoisomerase inhibitors such as camptothecinsand derivatives thereof, mitoxantrone, mitoguazone. In a preferredembodiment, the cytotoxin is selected from the group consisting ofnitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin, phenamet,phenesterine, prednimustine, trofosfamide, uracil mustard. In yetanother preferred embodiment, the cytotoxin is selected from the groupconsisting of nitrosoureas such as carmustine, flubenzuron, formoterol,lomustine, nimustine, ramustine. In one embodiment, the cytotoxin isselected from the group consisting of aziridines. In a preferredembodiment, the cytotoxin is selected from the group consisting ofbenzodopa, carboquone, meturedepa, and uredepa. In one embodiment, thecytotoxin is selected from the group consisting of an anti-tumorantibiotic. In a preferred embodiment, the cytotoxin is selected fromthe group consisting of enediyne antibiotics. In a more preferredembodiment, the cytotoxin is selected from the group consisting ofdynemicin, esperamicin, neocarzinostatin, and aclacinomycin. In anotherpreferred embodiment, the cytotoxin is selected from the groupconsisting of actinomycin, antramycin, bleomycins, actinomycin C,carabicin, carminomycin, and cardinophyllin, carminomycin, actinomycinD, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin,peplomycin, porfiromycin, puromycin, ferric adriamycin, rodorubicin,rufocromomycin, streptozocin, zinostatin, zorubicin. In yet anotherpreferred embodiment, the cytotoxin is selected from the groupconsisting of trichothecene. In a more preferred embodiment, thecytotoxin is selected from the group consisting of T-2 toxin, verracurinA, bacillocporin A, and anguidine. In one embodiment, the cytotoxin isan anti-tumor amino acid derivative. In a preferred embodiment, thecytotoxin is selected from the group consisting of ubenimex, azaserine,6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin isselected from the group consisting of folic acid analogues. In apreferred embodiment, the cytotoxin is selected from the groupconsisting of dimethyl folic acid, methotrexate, pteropterin,trimetrexate, and edatrexate. In one embodiment, the cytotoxin isselected from the group consisting of purine analogues. In a preferredembodiment, the cytotoxin is selected from the group consisting offludarabine, 6-mercaptopurine, tiamiprine, thioguanine. In yet anotherembodiment, the cytotoxin is selected from pyrimidine analogues. In apreferred embodiment, the cytotoxin is selected from the groupconsisting of ancitabine, gemcitabine, enocitabine, azacitidine,6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,floxuridine. In one embodiment, the cytotoxin is selected from the groupconsisting of androgens. In a preferred embodiment, the cytotoxin isselected from the group consisting of calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone. In anotherembodiment, the cytotoxin is selected from the group consisting ofanti-adrenals. In a preferred embodiment, the cytotoxin is selected fromthe group consisting of aminoglutethimide, mitotane, and trilostane. Inone embodiment, the cytotoxin is selected from the group consisting ofanti-androgens. In a preferred embodiment, the cytotoxin is selectedfrom the group consisting of flutamide, nilutamide, bicalutamide,leuprorelin acetate, and goserelin. In yet another embodiment, thecytotoxin is selected from the group consisting of a protein kinaseinhibitor and a proteasome inhibitor. In a particularly embodiment, thecytotoxin is selected from the group consisting of vinblastines,colchicines, taxanes, auristatins, and maytansinoids. In a particularembodiment, the cytotoxin is an auristatin, such as MMAE (monomethylauristatin E), MMAF (monomethyl auristatin F), MMAD (monomethylauristatin D) and the like. The synthesis and structure of auristatincompounds are described in US20060229253, the entire disclosure of whichis incorporated herein by reference.

The payload contains a reactive group which can react with the reactivegroup in the compound of formula (V) and thus covalently couple thepayload with the compound of formula (V). Compounds that do not containreactive groups require appropriate derivatization to give the payload.In one embodiment, the reactive group in the payload is maleimide, andthe compound without maleimide may be subjected to suitable reaction(s)to give a maleimide derivative. For example, MMAF is derivatized to givemc-MMAF (mc is maleimidocaproyl). MMAE is derivatized to givemc-Val-Cit-PAB-MMAE. mc in the above structures can be replaced by mcc(4-(maleimidomethyl)cyclohexane-1-carbonyl) or maleimide-R structure,wherein R is a C₁₋₂₀ alkylene, and optionally one or more (—CH₂—)structures in the alkylene can be replaced by —O—.

In an embodiment, step (a) comprises: obtaining System 2 throughreacting the linker of formula (V) with the payload. Preferably, thecompound of formula (V) is each independently covalently linked via areactive group comprised by moiety A1 or A2 to another reactive groupcomprised by the payload, forming the linker-payload intermediate offormula (IV).

The reactive groups comprised by moiety A1 or A2 are as described above.

In a particular embodiment, the covalently link formed between thecompound of formula (V) and the payload is one or more bonds selectedfrom amide bonds, disulfide bonds, thioether bonds, thioester bonds,peptide bonds, hydrazone bonds, ester bonds, ether bonds and urethanebonds.

In an embodiment, the reactive group in A1 or A2 in the compound offormula (V) is a maleimide or maleimide derivative, and the otherreactive group in the payload is a Michael acceptor. And after thereaction with the payload, the maleimide or maleimide derivative turnsinto a succinimide or succinimide derivative.

In an embodiment, p=0, q=1, the intermediate of formula (IV) is as shownin the following formula (IV-1):

D1-Y-Lk-(W-A2-P)_(t)  (IV-1)

In another embodiment, p=1, q=0, the structure of the compound offormula (IV) is as shown in the following formula (IV-2):

(P-A1-Y)_(t)-Lk-WD2  (IV-2)

In an embodiment, the linker-payload intermediate of formula (IV) whichcontains a succinimide or succinimide derivative may be subjected toring-opening reaction, so as to obtain a “ring-open” intermediate. Thering-opening reaction could be performed through a method similar tothat described in WO2015165413A. The ring-open intermediates are alsoincluded in the scope of formula (IV).

In an embodiment, the ring-opening reaction of the succinimide in thelinker-payload intermediate of formula (IV) forms ring-open intermediateas shown in the following formula (IV-1-2). Formula (IV-1-2) falls inthe scope of formula (IV-1).

D1-Y-Lk-(W-A2open-P)_(t)  (IV-1-2)

In another embodiment, the ring-opening reaction of the succinimide ringin the linker-payload intermediate of formula (IV) forms ring-openintermediate as shown in the following formula (IV-2-2). Formula(IV-2-2) falls in the scope of formula (IV-2).

(P-A1open-Y)_(t)-Lk-W-D2  (IV-2-2)

Wherein, “-A1open-” and “-A2open-” has a structure selected from

Specific Embodiments for the Linker-Payload Intermediate

In one embodiment, A1 and A2 are each independently selected from mc andmcc, and the maleimide functional group contained therein is linked to athiol group in the payload (P), and the linker and the payload arethereby connected to each other through a thiosuccinimide structure(thiosuccinimide linkage). The succinimide ring in the thiosuccinimidelinkage may be subjected to ring-opening reaction as described above,and obtain a ring-open thiosuccinimide structure

In an embodiment, the structure of L is as defined in formula (V-1-1),and the linker-payload intermediate of formula (IV-1) has the structureof formula (IV-1-1). The linker-payload intermediate of formula (IV-1-1)may be prepared through reaction of the corresponding linker of formula(V-1-1) with the payload (P).

In a specific embodiment, in formula (IV-1), D1 is G_(n), G is glycine,A2 is

Lk is L₄, L₄ is an optionally derivatized lysine, D2 is G_(n), G isglycine, and the linker-payload intermediate has the structure as shownin the following formula (IV-1-1):

In a preferred embodiment, in formula (IV-1-1), Y and W are both absent,the payload is mc(ring open)-Toxin, and the linker-payload intermediatehas the structure as shown in the following formula (IV-1-2) or formula(IV-1-2′):

wherein Toxin represents a cytotoxin as defined in formula (III); n, Lkand x are as defined in formula (IV-1-1), respectively.

In a more preferred embodiment, in formula (IV-1-2) and formula(IV-1-2′), the cytotoxin is MMAF, i.e., the payload is mc(ringopen)-MMAF, and the linker-payload intermediate has the structure asshown in the following formula (IV-1-3) or formula (IV-1-3′):

Formulae (IV-1-3) and (IV-1-3′) are isomers, wherein:

T, n, Lk and x are as defined in formula (IV-1-1), respectively.

In another specific embodiment, in formulae (IV-1-3) and (IV-1-3′), Lkis L₁-L₂-L₃, L₁ is —NH—, and L₃ is —(CO)—, L₂ is —(C₂H₄—O)_(i)—C₂H₄—,i=4, and the linker-payload intermediate has the structure as shown inthe following formula (IV-1-4) and (IV-1-4′):

Formulae (IV-1-4) and (IV-1-4′) are isomers, wherein:

n and x are as defined in formula (IV-1-1), respectively.

In a specific embodiment, in formula (IV-1), W does not exist, t is 1,the payload is Toxin, A1 is mcc, Lk is L₄, L₄ is an optionallyderivatized lysine, D2 is G_(n), G is glycine, and the linker-payloadintermediate has the structure as shown in the following formula(IV-1-5-1):

wherein Toxin, n, Y and x are as defined above.

In one embodiment, the linker-payload intermediate has the structure asshown in the following formula (IV-1-5) or (IV-1-5′):

wherein Toxin, n, Y and x are as defined above.

The linker-payload intermediate of formulae (IV-1-5) and (IV-1-5′) maybe prepared through ring-opening reaction of formula (IV-1-5-1).

In a preferred embodiment, the Toxin is a maytansinoid, preferably DM1.

In a specific embodiment, in formulae (IV-1-5) and (IV-1-5′), thecytotoxin is DM1, Y does not exist, and the linker-payload intermediatehas the structure as shown in the following formula (IV-1-6) or(IV-1-6′). Formula (IV-1-6) falls in the scope of formula (IV-1-5), andformula (IV-1-6′) falls in the scope of formula (IV-1-5′).

wherein n and x are as defined above.

Specific Embodiments for the Conjugate

In an embodiment, t is 1, and the conjugate has the structure selectedfrom the following formulas (1), (2), (2′), (3), (3′), (4), (4′), (5),(5′), (6), (6′).

In an embodiment, the structure of L-P is as defined in formula (V-1-1),and the conjugate of formula (III) has the structure of formula (1). Theconjugate of formula (1) may be prepared through conjugation reaction ofthe corresponding linker-payload intermediate of formula (V-1-1) withthe biomolecule (T).

wherein n is an integer of 3 to 10;

x is OH, NH₂ or Gly;

Lk is L₁-L₂-L₃;

T, Payload and z are as defined in formula (III), respectively; L₁, L₂,L₃, Y and W are as defined in formula (V), respectively.

In an embodiment, the structure of L-P is as defined in formulae(IV-1-2) or (IV-1-2′), and the conjugate of formula (III) has thestructure selected from the following formulae (2) and (2′). In aspecific embodiment, the structure of L-P is as defined in formulae(IV-1-3) or (IV-1-3′), and the conjugate of formula (III) has thestructure selected from the following formulae (3) and (3′). In a morespecific embodiment, the structure of L-P is as defined in formulae(IV-1-4) or (IV-1-4′), and the conjugate of formula (III) has thestructure selected from the following formulae (4) and (4′). Formula (4)falls in the scope of formula (3), formula (3) falls in the scope offormula (2), and formula (2) falls in the scope of formula (1). Formula(4′) falls in the scope of formula (3′), formula (3′) falls in the scopeof formula (2′), and formula (2′) falls in the scope of formula (1′).

In a preferred embodiment, Y and W are both absent, the payload informula (1) is mc(ring open)-toxin, and the structure of the conjugateis as shown in the following formula (2) or formula (2′):

wherein Toxin represents a cytotoxin as defined in formula (III); T, n,Lk, x and z are as defined in formula (1), respectively.

In a more preferred embodiment, the cytotoxin in formula (2) and formula(2′) is MMAF, i.e., the payload is mc(ring open)-MMAF, and the structureof the conjugate is as shown in the following formula (3) or formula(3′):

Formulae (3) and (3′) are isomers, wherein:

T, n, Lk, x and z are as defined in formula (1), respectively.

In another specific embodiment, Lk is L₁-L₂-L₃, L₁ is —NH—, and L₃ is—(CO)—, L₂ is —(C₂H₄—O)_(i)—C₂H₄—, i=4. The structure of the conjugateis as shown in the following formula (4) and (4′):

Formulae (4) and (4′) are isomers, wherein:

T, n, x and z are as defined in formula (1), respectively.

In a particular embodiment, the targeting molecule is the antibodyPertuzumab, hrS7, or MAAA1181a.

In an embodiment, t is 1, the structure of L-P is as defined in formulae(IV-1-5) or (IV-1-5′), and the conjugate of formula (III) has thestructure selected from the following formulae (5) and (5′). Theconjugate of (5) or (5′) may be prepared through conjugation reaction ofthe corresponding linker-payload intermediate of formula (IV-1-5) or(IV-1-5′) with the biomolecule (T). In a specific embodiment, theconjugate of formula (III) has the structure selected from the followingformulae (6) and (6′). The conjugate of (6) or (6′) may be preparedthrough conjugation reaction of the corresponding linker-payloadintermediate of formula (IV-1-6) or (IV-1-6′) with the biomolecule (T).Formula (6) falls in the scope of formula (5), and formula (6′) falls inthe scope of formula (5′).

In a specific embodiment, Y does not exist. The structure of theconjugate is as shown in the following formula (6) and (6′):

Formulae (6) and (6′) are isomers. In one embodiment, T is an anti-humanHER2 antibody. In another embodiment, T is a modified Trastuzumab.

Specific Embodiments for the Process

The process described herein is different from conventional chemicalcoupling processes in the art in that the conjugation step is catalyzedby a ligase in a site-specific manner, in which the ligase specificallyrecognizes the recognition motifs on the moieties to be conjugated.While in a conventional chemical coupling reaction, it is desirable topurify the moieties to be conjugated prior to the coupling reaction toavoid undesirable by-products resulted from non-specific coupling.

Therefore, in one aspect, the process according to the presentdisclosure obviates the need to purify the moieties to be conjugatedprior to the conjugation step, thereby reducing the overall operationaltime and steps while increasing the final yield. Accordingly, in someembodiments, System 1 and/or System 2 in step (a) further comprise oneor more impurities.

In another aspect, the process is particularly beneficial for conjugatescomprising chemically labile molecules, such as biomolecules (such asproteins). In a specific aspect, the process is suitable for thepreparation of a bioconjugate, wherein at least one of the first moietyand second moiety comprises a biomolecule.

Typically, processes for the manufacture of biomolecules involves cellculture methods, e.g., using either mammalian or bacterial host celllines to produce a target protein, such as an antibody or an antibodyfragment. In most cases, the harvest collection is subjected toclarification to remove cells and cellular debris, obtaining theharvested clarified cell culture fluid (HCCF), which contains impuritiessuch as, e.g., host cell proteins (HCP), media components and nucleicacids. In a conventional chemical coupling based process, the HCCF isfurther subjected to a series of purification steps to obtain a highpurity biomolecule for the downstream conjugation reaction. In someother cases, the harvest collection is subjected to extraction,clarification, concentration to precipitate the target protein, and thepellets are re-dissolved then subjected to purification. As the ligasein the process according to the present disclosure can specificallycatalyze conjugation between the first moiety and second moiety,impurities in System 1 and/or System 2 can hardly affect the conjugationefficiency and/or specificity in step (b), a high purity of System 1 andSystem 2 is no longer a prerequisite for step (b). In one embodiment, atleast one of System 1 and System 2 according to step (a) is a harvestedclarified cell culture fluid (HCCF). The HCCF can be obtained from atissue culture, a mammalian cell culture, a yeast cell culture, abacterial cell culture, a bacteriophage culture, etc. In someembodiments, other samples other than HCCF can also be used.

In another preferable aspect, the process according to the presentdisclosure can be flexibly integrated with the production process of abiomolecule to obtain a bioconjugate comprising the same. For examples,the process can be easily integrated with the production procedure ofmonoclonal antibodies or antibody fragments to produce bioconjugatescomprising the same. The bioconjugate can be manufactured by the samemanufacturing facility, in the same product cycle of the biomoleculecomprised therein, with a similar overall yield, while the originalmanufacturing facility and pipeline may remain largely unchanged.

In another embodiment, the process further comprises the steps of

(1) subjecting System 1 in step (a) before step (b), and/or

(2) subjecting System 2 in step (a) before step (b), and/or

(3) subjecting the conjugate obtained in step (b),

to one or more chromatography steps to remove one or more impurities.

The chromatography step can be independently selected from the groupconsisting of affinity chromatography, hydrophobic interactionchromatography, ion exchange chromatography, mixed mode chromatography,hydroxyapatite chromatography and a combination thereof. The ionexchange chromatography can be selected from the group consisting ofanion exchange chromatography, cation exchange chromatography, mixedmode ion exchange chromatography and a combination thereof. In apreferable embodiment, the ion exchange chromatography is a combinationof anion exchange chromatography and cation exchange chromatography. Insome embodiments, the chromatography steps in step (3) are also referredto as polishing steps. A polishing step may comprise affinitychromatography, hydrophobic interaction chromatography, anion exchangechromatography, cation exchange chromatography, mixed modechromatography and hydroxyapatite chromatography.

In one embodiment, at least one of the first moiety and second moietycomprises an antibody or an antibody fragment, and at least one of steps(1)-(3) comprises an affinity chromatography. The antibody can be aconventional antibody, a recombinant antibody, a multispecific antibody,a fully human antibody, a non-human antibody, a humanized antibody, achimeric antibody, an intrabody or a nanobody. The antibody can be anytype (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g. IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a andIgG2b), or any derivatives thereof. The antibody fragment can be an Fvfragment, an scFv fragment, a dsFv fragment, an scdsFv fragment, an Fdfragment, an Fab fragment, an scFab fragment, an Fab′ fragment, anF(ab′)₂ fragment, an Fc fragment or a diabody. Depending on the natureof the antibody or the antibody fragment, the affinity chromatographycan be Protein A affinity chromatography, Protein G affinitychromatography, Protein L affinity chromatography (such as Capto™Laffinity chromatography), KappaSelect affinity chromatography,LamdaFabSelect affinity chromatography or Mabselect™ affinitychromatography. In some embodiments, at least one of the first moietyand second moiety comprises an Fc fragment, and the affinitychromatography is Protein A affinity chromatography. In someembodiments, the first moiety comprises an Fc fragment. Preferably, theFc fragment is an Fc fragment of IgG-type antibodies, such as IgG1,IgG2, IgG3 and IgG4. In a particular embodiment, T is an antibody, andthe affinity chromatography is Protein A affinity chromatography. Thoseof skills in the art are able to select a suitable affinitychromatography approach based on the nature of the moieties to beconjugated.

In a preferable embodiment, the chromatography steps in steps (1), (2)and (3) are selected from affinity chromatography, ion exchangechromatography, and a combination thereof.

In one embodiment, steps (1) and (2) do not exist; and step (3) is acombination of affinity chromatography and ion exchange chromatography(Process 1). In another embodiment, at least one of step (1) and step(2) comprises an affinity chromatography, and step (3) comprises an ionexchange chromatography (Process 2). In yet another embodiment, at leastone of step (1) and step (2) comprises an affinity chromatography, andstep (3) comprises a combination of affinity chromatography and ionexchange chromatography

(Process 3). In yet another embodiment, at least one of step (1) andstep (2) comprises an affinity chromatography and/or an ion exchangechromatography, and step (3) comprises a combination of affinitychromatography, hydrophobic interaction chromatography and ion exchangechromatography (Process 4).

In a particular embodiment, steps (1) and (2) do not exist; and step (3)comprises the steps in any order:

(3a-1): Protein A affinity chromatography;

(3a-2): anion exchange chromatography; and

(3a-3): cation exchange chromatography.

In another particular embodiment, at least one of steps (1) and (2)comprises a

Protein A affinity chromatography, and step (3) comprises the steps inany order:

(3b-1): anion exchange chromatography; and

(3b-2): cation exchange chromatography.

In yet another particular embodiment, at least one of steps (1) and (2)comprises a Protein A affinity chromatography, and step (3) comprisesthe steps in any order:

(3c-1): Protein A affinity chromatography;

(3c-2): anion exchange chromatography; and

(3c-3): cation exchange chromatography.

In a particular embodiment, the first moiety and/or the second moietycomprises an Fc fragment, steps (1) and (2) do not exist, and step (3)comprises the steps in any order:

(3a-1): Protein A affinity chromatography;

(3a-2): anion exchange chromatography; and

(3a-3): cation exchange chromatography.

In another particular embodiment, the first moiety comprises an Fcfragment, step (1) comprises a Protein A affinity chromatography, andstep (3) comprises the steps in any order:

(3b-1): anion exchange chromatography; and

(3b-2): cation exchange chromatography.

In yet another particular embodiment, the first moiety comprises an Fcfragment, step (1) comprises a Protein A affinity chromatography, andstep (3) comprises the steps in any order:

(3c-1): Protein A affinity chromatography;

(3c-2): anion exchange chromatography; and (3c-3): cation exchangechromatography.

In yet another particular embodiment, the second moiety comprises an Fcfragment, step (2) comprises a Protein A affinity chromatography, andstep (3) comprises the steps in any order:

(3b-1): anion exchange chromatography; and

(3b-2): cation exchange chromatography.

In yet another particular embodiment, the second moiety comprises an Fcfragment, step (2) comprises a Protein A affinity chromatography, andstep (3) comprises the steps in any order:

(3c-1): Protein A affinity chromatography;

(3c-2): anion exchange chromatography; and

(3c-3): cation exchange chromatography.

In yet another particular embodiment, step (1) or step (2) comprises aProtein A affinity chromatography and an anion exchange chromatography,and step (3) comprises the steps in any order:

(3d-1): Protein A affinity chromatography;

(3d-2): cation exchange chromatography; and

(3d-3): hydrophobic interaction chromatography.

In one embodiment, the affinity chromatography is performed inbind-and-elute mode. In another embodiment, the ion exchangechromatography is performed in bind-and-elute mode or flow-through mode.

In a preferable embodiment, the anion exchange chromatography isperformed in flow-through mode. In one embodiment, the sample obtainedin a flow-through purification process step flows continuously into thenext process step. In another preferable embodiment, the cation exchangechromatography is performed in bind-and-elute mode. In one embodiment,the sample obtained in a bind-and-elute purification process step flowscontinuously into the next process step.

In one embodiment, step (b) is performed in batch mode, semi-continuousmode or continuous mode. In another embodiment, at least one of steps(a), (b) and (1) to (3) is performed in semi-continuous mode orcontinuous mode; preferably, steps (a), (b) and (1) to (3) are performedin continuous mode.

In a particular embodiment, the process of the present disclosure isperformed in continuous mode; the process comprising:

(a′): providing System 1 in fluid; and providing System 2 in fluid;

(b′): subjecting System 1 and/or System 2 independently tochromatography step to obtain an eluate of System 1 and/or an eluate ofSystem 2, wherein the eluate of System 1 and/or the eluate of System 2has reduced level of impurities;

(c′): mixing System 1 and System 2 in step (a′) or (b′) to form areaction fluid, and applying the ligase unit to the reaction fluid tocatalyze the conjugation reaction of T and the linker-payloadintermediate of formula (IV) and thereby obtaining a crude conjugatemixture, wherein the crude conjugate mixture comprises the targetconjugate and one or more impurities;

(d′): subjecting the crude conjugate mixture of step (c′) tochromatography step to remove the impurities, and obtaining the targetconjugate with desired purity;

wherein

steps (a′) to (d′) are connected to be in fluid communication with eachother, such that a sample can flow continuously from one process step tothe next.

The process according to the present disclosure can be adapted for thepreparation of a particular conjugate, for example, by furthercomprising additional steps selected from the group consisting offermentation, clarification, chromatography, pH adjustment, virusinactivation, virus filtration, ultrafiltration, diafiltration, sterilefiltration, formulation and a combination thereof. Those of skills inthe art will be able to combine such additional steps with the processaccording to the present disclosure, as well as arrange the sequentialorders of the steps for the preparation of a certain conjugate.

In an embodiment, the process of the present disclosure furthercomprises steps of virus inactivation, virus filtration, UF/DF and/orformulation. In an embodiment, the virus inactivation is done throughlow-pH treatment, for example, after Protein A affinity chromatography.Virus filtration can be performed after or before the conjugation step,i.e., step (b). Preferably, virus filtration is performed after at leastone chromatography step. In some embodiments, virus filtration isperformed after the conjugation step, for example, after the polishingsteps (for example, ADC Processes 1-3). In some other embodiments, virusfiltration is performed before the conjugation step, for example, afteran ion exchange chromatography (for example, ADC Process 4). In oneembodiment, UF/DF is performed after the conjugation step and beforestep (3) (for example, ADC Processes 3-4).

In a particular embodiment, step (1) or step (2) comprises a Protein Aaffinity chromatography, UF/DF is performed after the conjugation stepand before step (3), wherein step (3) comprises the steps in any order:

(3e-1): anion exchange chromatography; and

(3e-2): cation exchange chromatography.

In another particular embodiment, step (1) or step (2) comprises aProtein A affinity chromatography and an anion exchange chromatography,UF/DF is performed after the conjugation step and before step (3),wherein step (3) comprises cation exchange chromatography or hydrophobicinteraction chromatography.

In a particular embodiment, the conjugate is an ADC, and the process isas illustrated in FIG. 15 , including ADC Process 1, ADC Process 2, ADCProcess 3 and ADC Process 4. Comparing to the conventional ADC process,the process according to the present disclosure involves fewer steps,thereby reducing the operational time, materials and space for theprocess. Furthermore, in the conventional ADC process, conjugation istypically conducted after virus filtration; while in the processesaccording to the present disclosure, conjugation can be performed before(Processes 1-3) or after (Process 4) virus filtration, giving moremanufacturing flexibility.

In an embodiment, the disclosure provides a ligase fusion protein (thepresent ligase fusion protein) comprising a ligase and a Halo tag. Forexample, the disclosure provides

-   1.1. The present ligase fusion protein wherein the ligase is a    transpeptidase, preferably a sortase.-   1.2. The present ligase fusion protein wherein the ligase is a    sortase A.-   1.3. Any foregoing ligase fusion protein wherein the ligase is a    sortase, preferably a sortase A; and/or the recognition motif of the    ligase donor substrate is LPXTGJ; preferably LPXTG or LPETGG; and/or    the recognition motif of the ligase acceptor substrate is G_(n),    wherein G is glycine (Gly), and n is an integer of 3-10; X is any    natural or unnatural amino acid; J is absent, or is an amino acid    fragment comprising 1-10 amino acids, wherein each amino acid is    independently any natural or unnatural amino acid; preferably, J is    absent or is G_(m), wherein m is an integer of 1-10.-   1.4. Any foregoing ligase fusion protein wherein the ligase is    capable of catalyzing the conjugation between a first moiety    comprising the recognition motif of the ligase donor substrate (e.g.    comprising the terminal sequence LPXTG or LPXTGG, wherein X is any    natural amino acid) and a second moiety comprising the recognition    motif of the ligase acceptor substrate (e.g., comprising a terminal    polyglycine sequence, e.g., GGG) to produce a conjugate of the first    moiety and the second moiety.-   1.5. Any foregoing ligase fusion protein wherein the ligase    comprises an amino acid sequence selected from the group consisting    of    -   a. any of SEQ ID NOs: 1-26;    -   b. any of SEQ ID NOs: 1-26 wherein the amino acid residues at        positions 34, 100, 105 and 136 are optionally substituted with        Ser, Asn, Ala and Thr (i.e., [Ser34][Asn100][Ala105][Thr136],        SNAT), Tyr, Asn, Ala and Thr (i.e.,        [Tyr34][Asn100][Ala105][Thr136], YNAT), Trp, Asn, Asp and Thr        (i.e., [Trp34][Asn100][Asp105][Thr136], WNDT), or Val, Asn, Asn        and Ser (i.e., [Val34][Asn100][Asn105][Ser136], VNNS),        respectively; for example wherein the ligase comprises the amino        acid sequence of SEQ ID NO: 27 (i.e., the SNAT counterpart of        SEQ ID NO: 1); and    -   c. an amino acid sequence having sortase activity and a sequence        identity of at least about 85%, at least about 90%, at least        about 95%, or at least about 99%, to any of (a) or (b).-   1.6. Any foregoing ligase fusion protein wherein the Halo tag is a    polypeptide that catalyzes a removal of the halogen from a haloalkyl    moiety to form a covalent bond with the dehalogenated alkyl moiety.-   1.7. Any foregoing ligase fusion protein wherein the Halo tag is    derived from a bacterial haloalkane dehalogenase that catalyzes a    removal of the halogen from a haloalkyl moiety to form a covalent    bond with the dehalogenated alkyl moiety and which is mutated to    prevent hydrolysis of the covalent bond thus formed, e.g., a    haloalkane dehalogenase from Xanthobacter autotrophicus or    Rhodococcus rhodochrous wherein a residue involved in hydrolysis is    mutated, e.g., wherein a histidine residue at a position    corresponding to amino acid residue 272 of a Rhodococcus rhodochrous    dehalogenase is mutated.-   1.8. Any foregoing ligase fusion protein wherein the Halo tag    comprises the amino acid sequence of SEQ ID NO: 28; or an amino acid    sequence having dehalogenase activity and a sequence identity of at    least about 85%, at least about 90%, at least about 95%, or at least    about 99% to SEQ ID NO: 28.-   1.9. Any foregoing ligase fusion protein wherein the ligase has an    isoelectric point (pI) of about 7.5 to about 10.0; the Halo tag has    an isoelectric point of about 4.5 to about 5.0, and the isoelectric    point (pI) of the ligase fusion protein is about 2.0 to about 4.5 pH    units lower than that of the ligase.-   1.10. Any foregoing ligase fusion protein comprising the sequence of    SEQ ID NO: 29; or an amino acid sequence having dehalogenase    activity, sortase activity, and a sequence identity of at least    about 85%, at least about 90%, at least about 95%, or at least about    99% to SEQ ID NO: 29.

In another embodiment, the disclosure provides an immobilized ligase(the present immobilized ligase), comprising a ligase linked via a Halotag to a support, e.g., wherein the ligase is immobilized by thereaction of a ligase fusion protein comprising a ligase and a Halo tag,e.g., any of the present ligase fusion proteins, with a supportcomprising haloalkyl linkers, preferably chloroalkyl linkers, on itssurface, such that the ligase fusion protein is immobilized on thesupport through covalent interaction between the haloalkyl linker andthe Halo tag; for example,

-   1.1. The present immobilized ligase wherein the ligase is    immobilized by the reaction of a ligase fusion protein comprising a    ligase and a Halo tag with a support comprising haloalkyl linkers,    wherein the ligase fusion protein is any of the present ligase    fusion proteins.-   1.2. Any foregoing immobilized ligase wherein the ligase is    immobilized by the reaction of a ligase fusion protein comprising a    ligase and a Halo tag with a support comprising a haloalkyl linker,    wherein the haloalkyl linker is produced by a haloalkyl substrate    having the structure of formula (I-1-1) or (I-1):

-   -   wherein u is an integer of 1-20, v is an integer of 0-20, and w        is an integer of 1 to 19.

-   1.3. Any foregoing immobilized ligase, wherein the ligase is    immobilized by the reaction of a ligase fusion protein comprising a    ligase and a Halo tag with a support comprising a haloalkyl linker,    e.g., wherein the support has the structure of formula (II-1) or    (II):

-   -   wherein the

-   -    is a support, e.g. selected from resin, a bead, a membrane, a        gel, a matrix, a film, a plate, a well, a tube, a glass slide or        a surface, preferably a resin, more preferable an agarose resin,        a silicone resin, a polymethyl methacrylate resin or cellulose        resin, most preferably a highly crosslinked agarose resin; and    -   wherein u is an integer of 1-20, v is an integer of 0-20, and w        is an integer of 1 to 19.    -   [Note that for the sake of clarity, only a single        chloroalkyl-linker moiety is depicted attached to the support,        but it is understood that there would be many such        chloroalkyl-linker moieties attached to the support.]

-   1.4. Any foregoing immobilized ligase having the following    structure:    -   Support ---- Linker ---- HaloTag ---- Ligase    -   wherein    -   Support is a solid support, e.g. selected from resin, a bead, a        membrane, a gel, a matrix, a film, a plate, a well, a tube, a        glass slide or a surface, preferably a resin, more preferable an        agarose resin, a silicone resin, a polymethyl methacrylate resin        or cellulose resin, most preferably a highly crosslinked agarose        resin;    -   Linker is a linker moiety, covalently bound to the Support,        e.g., comprising a chain of 10 to 60 carbon atoms, optionally        comprising one or more ether, ester, carbamate, and/or amide        bonds, e.g., a linker moiety of Formula (II-1′) or (II′)

-   -   wherein u is an integer of 1-20, v is an integer of 0-20, and w        is an integer of 1 to 19;    -   HaloTag is a Halo tag (haloalkane dehalogenase polypeptide),        covalently bound to the linker;    -   Ligase is a ligase polypeptide;    -   wherein one or more “---- Linker ---- HaloTag ---- Ligase”        moieties are bound to the same Support.

-   1.5. Any foregoing immobilized ligase wherein the ligase is a    sortase, preferably a sortase A; and/or the recognition motif of the    ligase donor substrate is LPXTGJ; preferably LPXTG or LPETGG; and/or    the recognition motif of the ligase acceptor substrate is G_(n),    wherein G is glycine (Gly), and n is an integer of 3-10; X is any    natural or unnatural amino acid; J is absent, or is an amino acid    fragment comprising 1-10 amino acids, wherein each amino acid is    independently any natural or unnatural amino acid; preferably, J is    absent or is G_(m), wherein m is an integer of 1-10.

-   1.6. Any foregoing immobilized ligase wherein the ligase is capable    of catalyzing the conjugation between a first moiety comprising the    recognition motif of the ligase donor substrate (e.g. comprising the    terminal sequence LPXTG or LPXTGG, wherein X is any natural amino    acid) and a second moiety comprising the recognition motif of the    ligase acceptor substrate (e.g., comprising a terminal polyglycine    sequence, e.g., GGG) to produce a conjugate of the first moiety and    the second moiety.

-   1.7. Any foregoing immobilized ligase wherein the ligase comprises    an amino acid sequence selected from the group consisting of    -   a. any of SEQ ID NOs: 1-26;    -   b. any of SEQ ID NOs: 1-26 wherein the amino acid residues at        positions 34, 100, 105 and 136 are optionally substituted with        Ser, Asn, Ala and Thr (i.e., [Ser34][Asn100][Ala105][Thr136],        SNAT), Tyr, Asn, Ala and Thr (i.e.,        [Tyr34][Asn100][Ala105][Thr136], YNAT), Trp, Asn, Asp and Thr        (i.e., [Trp34][Asn100][Asp105][Thr136], WNDT), or Val, Asn, Asn        and Ser (i.e., [Val34][Asn100][Asn105][Ser136], VNNS),        respectively; for example wherein the ligase comprises the amino        acid sequence of SEQ ID NO: 27 (i.e., the SNAT counterpart of        SEQ ID NO: 1); and    -   c. an amino acid sequence having sortase activity and a sequence        identity of at least about 85%, at least about 90%, at least        about 95%, or at least about 99%, to any of (a) or (b).

-   1.8. Any foregoing immobilized ligase wherein the Halo tag is a    polypeptide that catalyzes a removal of the halogen from a haloalkyl    moiety to form a covalent bond with the dehalogenated alkyl moiety.

-   1.9. Any foregoing immobilized ligase wherein the Halo tag is    derived from a bacterial haloalkane dehalogenase that catalyzes a    removal of the halogen from a haloalkyl moiety to form a covalent    bond with the dehalogenated alkyl moiety and which is mutated to    prevent hydrolysis of the covalent bond thus formed, e.g., a    haloalkane dehalogenase from Xanthobacter or Rhodococcus wherein a    residue involved in hydrolysis is mutated, e.g., wherein a histidine    residue at a position corresponding to amino acid residue 272 of a    Rhodococcus dehalogenase is mutated.

-   1.10. Any foregoing immobilized ligase wherein the Halo tag    comprises the amino acid sequence of SEQ ID NO: 28; or an amino acid    sequence having dehalogenase activity and a sequence identity of at    least about 85%, at least about 90%, at least about 95%, or at least    about 99% to SEQ ID NO: 28.

-   1.11. Any foregoing immobilized ligase wherein the ligase has an    isoelectric point (pI) of about 7.5 to about 10.0; the Halo tag has    an isoelectric point of about 4.5 to about 5.0, and the isoelectric    point (pI) of the ligase fusion protein is about 2.0 to about 4.5 pH    units lower than that of the ligase.

-   1.12. Any foregoing immobilized ligase comprising the sequence of    SEQ ID NO: 29; or an amino acid sequence having dehalogenase    activity, sortase activity, and a sequence identity of at least    about 85%, at least about 90%, at least about 95%, or at least about    99% to SEQ ID NO: 29.

-   1.13. Any foregoing immobilized ligase comprising the sequence of    SEQ ID NO: 29; or an amino acid sequence having dehalogenase    activity, sortase activity, and a sequence identity of at least    about 85%, at least about 90%, at least about 95%, or at least about    99% to SEQ ID NO: 29, bound via a linker to a support.

In another embodiment, the disclosure provides a process (the presentprocess) for the preparation of a conjugate comprising a first moietyand a second moiety, e.g., a drug-antibody conjugate, wherein one of thefirst moiety and the second moiety comprises a recognition motif of theligase donor substrate, and the other one of the first moiety and thesecond moiety comprises the recognition motif of the ligase acceptorsubstrate, the process comprising contacting the first moiety with thesecond moiety in the presence of a ligase unit which is an immobilizedligase or a ligase fusion protein comprising a ligase and a Halo tag;for example,

-   1.1. The present process wherein the ligase unit is an immobilized    ligase comprising a ligase linked via a Halo tag to a support, e.g.,    wherein the immobilized ligase is any of the present immobilized    ligases.-   1.2. The present process wherein the ligase unit is any of the    present ligase fusion proteins.-   1.3. Any foregoing process comprising the steps of:    -   (a) providing System 1 comprising the first moiety and providing        System 2 comprising the second moiety; and    -   (b) contacting the ligase unit with System 1 and System 2 in        step (a) to catalyze the conjugation reaction between the first        moiety and the second moiety to obtain the conjugate;    -   wherein the first moiety and the second moiety each        independently comprises a biomolecule, a protein, an antibody,        an antibody fragment, a receptor, a signal transduction factor,        a cell growth factor, a nucleic acid or a nucleic acid analogue,        a small molecule compound, a glycan, a PEG moiety, a        radionuclide, a cytokine, an immunomodulator, a tracer molecule,        a fluorophore, a fluorescent molecule, a peptide, a polypeptide,        or a peptidomimetic; and    -   wherein one of the first moiety and the second moiety further        comprises the recognition motif of the ligase donor substrate,        and the other one of the first moiety and the second moiety        comprises the recognition motif of the ligase acceptor        substrate, such hat the ligase unit will catalyze conjugation        between the recognition motif of the ligase donor substrate and        the recognition motif of the ligase acceptor substrate;        -   for example, wherein step (b) is performed in batch mode,            semi-continuous mode or continuous mode.-   1.4. The foregoing process further comprising the steps of    -   (1) subjecting System 1 in step (a) before step (b), and/or    -   (2) subjecting System 2 in step (a) before step (b), and/or    -   (3) subjecting the conjugate obtained in step (b),    -   to one or more chromatography steps to remove one or more        impurities;        -   for example, wherein the chromatography step is            independently selected from affinity chromatography,            hydrophobic interaction chromatography, ion exchange            chromatography, and a combination thereof; wherein the ion            exchange chromatography is selected from anion exchange            chromatography, cation exchange chromatography, and a            combination thereof;        -   preferably, the chromatography step is selected from            affinity chromatography, ion exchange chromatography, and a            combination thereof;        -   for example, wherein at least one of steps (a), (b) and            (1)-(3) is performed in semi-continuous mode or continuous            mode; e.g., wherein, steps (a), (b) and (1)-(3) are            performed in continuous mode;        -   for example, wherein at least one of the first moiety and            the second moiety comprises an antibody or an antibody            fragment, and at least one of steps (1)-(3) comprises an            affinity chromatography; e.g., wherein the antibody or            antibody fragment comprises an Fc fragment, and the affinity            chromatography is Protein A affinity chromatography;        -   for example, wherein the first moiety or the second moiety            comprises an Fc fragment,        -   steps (1) and (2) do not exist, and step (3) comprises the            steps in any order:        -   (3a-1): Protein A affinity chromatography;        -   (3a-2): anion exchange chromatography; and        -   (3a-3): cation exchange chromatography;        -   or        -   step (1) or step (2) comprises a Protein A affinity            chromatography, and step (3) comprises the steps in any            order:        -   (3b-1): anion exchange chromatography; and        -   (3b-2): cation exchange chromatography;        -   or        -   step (1) or step (2) comprises a Protein A affinity            chromatography, and step (3) comprises the steps in any            order:        -   (3c-1): Protein A affinity chromatography;        -   (3c-2): anion exchange chromatography; and        -   (3c-3): cation exchange chromatography;        -   or        -   step (1) or step (2) comprises a Protein A affinity            chromatography and an anion exchange chromatography, and            step (3) comprises the steps in any order:        -   (3d-1): Protein A affinity chromatography;        -   (3d-2): cation exchange chromatography; and        -   (3d-3): hydrophobic interaction chromatography;        -   for example, wherein the affinity chromatography is            performed in bind-and-elute mode; and/or the ion exchange            chromatography is performed in bind-and-elute mode or            flow-through mode; e.g., wherein, the anion exchange            chromatography is performed in flow-through mode; and/or the            cation exchange chromatography is performed in            bind-and-elute mode.-   1.5. Any foregoing process wherein one or more impurities are    present in the reaction between the first moiety and the second    moiety.-   1.6. Any foregoing process wherein the first moiety or the second    moiety is comprised in a harvested clarified cell culture fluid    (HCCF).-   1.7. Any foregoing process wherein the ligase unit is a ligase    fusion protein comprising a ligase and a Halo tag, further    comprising the step of reacting the ligase unit with a support    comprising haloalkyl linkers and, after the reaction between the    first moiety and the second moiety is substantially completed,    removing the immobilized ligase, e.g., any of the present    immobilized ligases, thus formed.-   1.8. Any foregoing process wherein the ligase unit is an immobilized    ligase, e.g., any of the present immobilized ligases, further    comprising removing the immobilized ligase after the reaction    between the first moiety and the second moiety is substantially    completed.-   1.9. Any foregoing process wherein the conjugate has the structure    of formula (III), the first moiety comprises T, and the second    moiety comprises a linker-payload intermediate of formula (IV):

-   -   wherein    -   T comprises a biomolecule, which is optionally modified to have        one of the recognition motif of the ligase donor substrate and        the recognition motif of the ligase acceptor substrate;    -   L comprises a linker, which comprises the other of the        recognition motif of the ligase donor substrate and the        recognition motif of the ligase acceptor substrate;    -   P comprises a payload;    -   z is an integer of 1-20;    -   t is an integer of 1-20.

-   1.10. The foregoing process, wherein    -   T comprises a protein, a peptide, an antibody, an antibody        fragment, a receptor, a signal transduction factor, a cell        growth factor and a nucleic acid or analogue; and/or P comprises        hydrogen, a small molecule compound, preferably a toxin, a        glycan, a PEG moiety, a radionuclide, a cytokine, an        immunomodulator, a nucleic acid or analogue, a tracer molecule,        a fluorophore, a fluorescent molecule, a peptide, a polypeptide,        a peptidomimetic, an antibody, an antibody fragment, or a        protein.

-   1.11. Any foregoing process wherein the ligase is a sortase,    preferably a sortase A; and/or the recognition motif of the ligase    donor substrate is LPXTGJ; preferably LPXTG or LPETGG; and/or the    recognition motif of the ligase acceptor substrate is G_(n), wherein    G is glycine (Gly), and n is an integer of 3-10;    -   X is any natural or unnatural amino acid;    -   J is absent, or is an amino acid fragment comprising 1-10 amino        acids, wherein each amino acid is independently any natural or        unnatural amino acid; preferably, J is absent or is G_(m),        wherein m is an integer of 1-10.

Beneficial Effects

In an aspect, the present disclosure provides a ligase fusion proteinhaving at least one of the following advantageous features:

(1) the ligase fusion protein is highly expressible and soluble, therebylowing the cost for enzyme purification;

(2) the ligase fusion protein can be easily purified with a high purityand activity in large amount, therefore is particularly suitable forindustrial applications; and

(3) the ligase fusion protein can be easily immobilized at aphysiological condition, which not only benefits the storage andshipping of the ligase but also helps in preserving the maximumenzymatic activity and improving the manufacturing scalability.

In another aspect, provided is an immobilized ligase comprising theligase fusion protein. The immobilized ligase has at least one of thefollowing advantageous features:

(1) high stability;

(2) high reusability, the immobilized ligase can easily be retrieved andseparated from the reaction system after the reaction completes, whilethe enzymatic activity is substantially uncompromised;

(3) good alkali resistance, making alkali-based cleansing of theimmobilized ligase possible; and

(4) high enzyme capacity and high enzymatic activity, therefore, theimmobilized ligase can catalyze conjugation reactions with highlyconcentrated enzyme activity in a confined space, thereby saving workingand storage space and cost of regents comparing to the free ligase.

Therefore, the immobilized ligase according to the present disclosure iscontrollable, reusable, cost-efficient and easy to scale up, and thus isparticularly advantageous for industrial applications.

In a particular aspect, the ligase fusion protein has an alteredisoelectric point compared to the ligase from which the ligase fusionprotein is derived, allowing effective removal of carryover enzymecontaminants from the final conjugate products. This feature isespecially important for conjugates that do not comprise affinitymoieties for efficient affinity purification. For example, for aconjugate with a pI of about 8 to about 9, a ligase fusion protein witha pI of about 5 to about 6 can be used, and the conjugate and the ligasefusion protein can be separated with anion exchange chromatography, inwhich the ligase fusion protein binds to the chromatography media whilethe conjugate flows through, or with a cation exchange chromatography,in which the conjugate binds to the chromatography media while theligase fusion protein flows through. More importantly, owing to thealtered pI of the ligase fusion protein, possible trace amounts of freeenzymes (e.g., those non-specifically adsorbed on the support may falloff during catalysis) can be easily removed.

In another aspect, the present disclosure provides a process forpreparing a conjugate using the ligase fusion protein or the immobilizedligase according to the present disclosure.

Conventional processes for the preparation of conjugates containingprotein moieties include at least two sets of purification processes:the upstream and downstream purification of the protein before theconjugation step, and the downstream purification of the conjugationproduct after the conjugation step, each including severalchromatography steps. For the preparation of ADC (antibody-drugconjugate), the current mainstream conjugation technology is chemistrybased, wherein the drug is chemically conjugated to lysine or cysteineresidue in the antibody via a linker. Before the conjugation step,antibodies with a high purity is prepared through upstream anddownstream processes, since antibody with a lower purity would result inunpredictable outcomes, such as by-products resulting from the couplingof the payload and the lysine/cysteine in the impurities in the antibodyfeed. Impurities in the antibody feed decrease the conjugation stepyield, and thereby put pressure on the process productivity, causingrequirements to increase the input, which in turn aggravates thecomplexity of the whole process. After the conjugation step, anotherdownstream purification process is required to remove the aggregates,solvents, by-products and impurities from ADC. The dual downstream stepsin the conventional process significantly increase cost and time, andsimultaneously lower the yield. Moreover, the conjugation reaction hasto be conducted in chemical isolators for safety reasons, making theprocess difficult to scale up. Overall, the conventional processesinvolve multiple upstream and downstream purification steps, which aretime-consuming, uneconomic, inflexible and lack of scalability.

The process of the present disclosure achieves at least one of thefollowing technical effects:

(1) owing to the substrate specificity of the ligase, the process can becarried out without a prior purification procedure of the moieties to beconjugated from raw materials, thereby leading to reduced overalloperational time and steps, reduced cost (e.g., pure water or water forinjection, reagents for various buffers, chromatography media and thelike), and improved yield;

(2) the process can be flexibly integrated with the procedures of thebiomolecule moieties like antibodies to be conjugated, that is, thetarget conjugate can be manufactured by the same manufacturing facility,in the same product cycle of the biomolecule moieties comprised therein,with a similar overall yield, while the original manufacturing facilityand pipeline may remain largely unchanged;

(3) the process can be easily scaled up to meet industrial needs,especially when an immobilized ligase is used;

(4) simplified in-process product quality analysis is achieved;

(5) efficient removal of impurities such as excess reaction material,and residual enzyme contaminants carried over from the upstreamcatalytic reaction is realized; and

(6) improved space-time economy is achieved, etc.

In addition to the above advantages, the process of the presentdisclosure is particularly advantageous for the preparation of abioconjugate over the conventional process at least in the followingaspects:

(1) formation of aggregates (such as antibody and ADC aggregates) isminimum, thereby increasing the final yield while lessening the workloadfor aggregate removal; and

(2) the DAR ratio and conjugation site on the bioconjugate can be easilymanipulated, thereby producing bioconjugates with a higher homogeneityand well-defined physicochemical characteristics.

EXAMPLES

In order to illustrate the objects and technical solutions more clearly,the present disclosure is further described below with reference tospecific examples. It is to be understood that the examples are notintended to limit the scope of the disclosure. The specific experimentalmethods which are not mentioned in the following examples are carriedout according to conventional experimental methods.

Instruments, Materials and Reagents

Unless otherwise stated, the instruments and reagents are commerciallyavailable or can be prepared according to conventional means in the art.

MabSelect Sure ProA is obtained from GE; Q Sepharose FF/Capto S impactare obtained from GE. CHO cells for antibody expression are obtainedfrom Thermo fisher Scientific. pcDNA3.3 are obtained from LifeTechnology.

HIC-HPLC: Butyl-HIC; mobile phase A: 25 mM PB, 2M (NH₄)₂SO₄, pH 7.0;mobile phase B: 25 mM PB, pH 7.0; flow rate: 0.8 ml/min; acquisitiontime: 25 mM; injection amount: 20 μg; column temperature: 25° C.;detection wavelength: 280 nm; sample chamber temperature: 8° C.

General Procedures General Procedures for Antibody Production

The processes for antibody preparation can be found in e.g.US20170112944A1, which are fully incorporated by reference in itsentirety. Briefly, a plasmid construct encoding the anti-human HER2antibody Trastuzumab is transfected into CHO cells, wherein the antibodyis modified at the C-terminus of the light chain by comprising a donorrecognition motif LPETGG to obtain the antibody T-LCCT_(L)-HC.Transfected CHO cells are screened for a highly expressed cellpopulation, which is cultured with reference to the culture process ofTrastuzumab in a 5-10 L reactor. The cell culture is centrifuged toobtain a harvested clarified cell culture fluid (HCCF).

The HCCF can be used as the antibody feed for conjugation reaction(Process 1) or further subjected to downstream processing to provide apurified antibody for conjugation reaction (Processes 2 and 3).

General Procedures for Antibody Purification

The processes for antibody purification can be found in e.g.US20170112944A1, which are fully incorporated by reference in itsentirety. Briefly, the purification of T-LCCT_(L)-HC is carried out in astandard process using the combination of Protein A affinitychromatography (MabSelect Sure ProA) and Sepharose S cation exchangechromatography, the purified products are dissolved in the originalTrastuzumab drug buffer (5 mM histidine-HCl, 2% Trehalose, 0.009%Polysorbate 20, pH 6.0).

General Procedures for the Linker-Payload Intermediate Preparation

The processes for the production of linker-payload intermediate can befound in e.g. US20170112944A1, which are fully incorporated by referencein its entirety. Briefly, the procedures are:

A solution of the linker having the structure of formula (V-1-2) isincubated with a solution of the payload to form a linker-payloadintermediate having the formula of (IV-1-5-1), which is furthersubjected to ring-open reaction to obtain a ring-open linker-payloadintermediate having the structure of formula (IV-1-5) or (IV-1-5′). Inthe Examples, the payload is DM1, and the linker-payload intermediatehas the structure of formula (IV-1-6) or (IV-1-6′). The ring-openlinker-payload intermediate is purified and analyzed by HPLC.

General Procedures for the Conjugation Reaction

The processes for the production of ADCs using antibodies purified fromProtein A affinity chromatography can be found in e.g. US20170112944A1,which are fully incorporated by reference in its entirety. Briefly, theprocedures are:

Prepare buffers containing the antibody T-LCCT_(L)-HC (1-100 mg/ml) andthe linker-payload intermediate (0.1-50 mg/ml) having the structure offormula (IV-1-6) or (IV-1-6′) separately as described above.

Fill the immobilized Halo-Sortase in a container in a desired amount.Treat the immobilized Halo-Sortase with 20 mM Tris-HCl, 1-3 M NaCl (pH6.0-10.0), 0.1-1.0 M

NaOH. Pre-warm the immobilized Halo-Sortase at 10-40° C. for about 30min or above in an air bath or a water bath. Mix the antibody solutionand the linker-payload intermediate solution according to theestablished ratio (antibody:linker-payload intermediate=1:1-1:100) toobtain a mixture, and then add the mixture to a container containing thetreated immobilized Halo-Sortase (pull-down mode) or to the Halo-Sortasecolumn (flow-through mode). Start the conjugation reaction. The reactiontime is 5 minutes to 24 hours.

After the conjugation reaction is completed, collect the reactionsolution or the flow-through from the immobilized Halo-Sortase column toobtain a crude conjugate mixture comprising the target conjugate, whichhas the structure of formula (6) or (6′). Subject the crude conjugatemixture to HIC-HPLC for analysis of DAR of the ADC to determine theconjugation efficiency of the reaction.

General Procedures for the Protein A Affinity Chromatography

Equilibrate the column with 20 mM Tris, 150 mM NaCl, pH 7.5, and loadthe crude conjugate mixture. Continue to flush with 20 mM Tris, 150 mMNaCl, pH 7.5, until the desired offset (baseline) is reached.Optionally, wash the impurities with citric acid-sodium citrate buffer,pH 5.0 (Wash step). Elute the desired ADC with citric acid-sodiumcitrate buffer, pH 3.3-3.7. Collect the eluate containing the desiredADC. Adjust the pH of the eluate to pH 5.0-6.0 using 1 M Tris-HCl, pH9.0. Analyze the eluate for residual impurities, such as HCP, DNA, andProtein A, using ELISA analysis or qPCR.

General Procedures for the Anion Exchange Chromatography

Pack the column with Q Sepharose FF medium. Equilibrate the column with20-100 mM Tris-HCl pH 6.5-8.0, and load the combined eluate collectedfrom the Protein A affinity chromatography. Collect the flow-throughcontaining the target ADCs. Continue to flush with 20 mM Tris-HCl pH6.5-8.0, until the desired offset (baseline) is reached. Regenerate thecolumn with 20-100 mM Tris-HCl, 1 M NaCl pH 6.5-8.0. Conductclean-in-position (CIP) for 30 min using 1 M NaOH. Analyze the eluatefor residual impurities, such as HCP, DNA, and Protein A.

General Procedures for the Cation Exchange Chromatography

Pack the column with Capto S ImpAct medium. Equilibrate the column withcitric acid-sodium citrate buffer, pH 5.0-6.0, and load the eluate fromthe Protein A affinity chromatography. Continue to flush with citricacid-sodium citrate buffer, pH 5.0-6.0, until the desired offset(baseline) is reached. Elute the target ADCs with citrate-sodium citratebuffer, 100-500 mM NaCl, pH 5.0-6.0. Collect the eluate containing thetarget ADC. Regenerate the column with citrate-sodium citrate buffer, 1M NaCl, pH 6.0. Conduct clean-in-position (CIP) for 30 min using 1 MNaOH. Analyze the eluate for residual impurities, such as HCP, DNA, andProtein A.

Example 1: Preparation of the Ligase Fusion Protein 1.1 Cloning andPurification of SrtAs

Nucleic acids encoding the SrtA having an amino acid sequence selectedfrom SEQ ID NO: 1-26 and their variants([Ser34][Asn100][Ala105][Thr136], SNAT; [Tyr34][Asn100][Ala105][Thr136],YNAT; [Trp34][Asn100][Asp105][Thr136]; WNDT[Val34][Asn100][Asn105][Ser136], VNNS) were synthesized by genesynthesis standard method and sub-cloned into the expression vectorpET-21a(+) at NdeI and EcoRI by Gibson Assembly. And a HiS₆ tag wasinserted at the N-terminal of the SrtA open reading frame.

SrtA expression plasmids were transformed into E. coli BL21(DE3). UntilOD600=0.5-0.8 after culturing at 37° C. in LB with 50 μg/ml Ampicillin,IPTG was added to a final concentration of 0.2 mM and sortase expressionwas induced at 25° C. for 12 hours. The target cells were harvested bycentrifugation and re-suspended in lysis buffer (50 mM Tris pH 8.0, 300mM NaCl) and then lysed by sonication and the supernatant was purifiedon Ni-NTA agarose following the manufacturer's instructions. The purityas judged by SDS-PAGE of the sortases were >90%. SrtAs' concentrationwere calculated from the measured A280 using the extinction coefficientmethod.

1.2 Evaluation of Sortase Activities

Recombinant SrtAs as prepared in Example 1.1 were subjected to afluorometric assay to measure their sortase activities. Reactions (totalvolume 100 μL, 0.085 mM Abz-LPETGK-Dnp, 18 mM tri-glycine in buffer A(buffer A: 5 mM CaCl₂, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) in 96-wellplate were initiated at 37° C. by adding 0.625 μM purified SrtAs orvariants. Abz-LPETGK-Dnp is an internally quenched peptide with2-aminobenzimidazole (Abz) as the fluorophore and 2, 4-dinitrophenyl(Dnp) as the quencher. Upon cleavage of the peptide LPETGK by sortase,Dnp and Abz are separated and the fluorescent signal from Abz can bedetected to indicate the sortase activity. The increase in fluorescencesignal was continuously collected after 1 hr (λ_(exe)/λ_(em)=320 nm/420nm, Gain=85, Biotek Cytation3 plate reader).

Activities of exemplary SrtAs, SrtA derived from Staphylococcus warneri(SEQ ID NO: 3) and its variant ([Ser34][Asn100][Ala105][Thr136], SNAT),are shown in bar graphs (FIG. 1 ). The result shows that the SNATvariant SrtA has about 1-fold higher activity than its wild type SrtA(SEQ ID NO: 3), while both of them have sortase activity.

1.3 Cloning of the Ligase Fusion Protein According to the PresentDisclosure (Halo-Sortase)

Nucleic acids encoding the ligase fusion proteins according to thepresent disclosure, each comprises a SrtA having an amino acid sequenceselected from SEQ ID NO: 1-26 and their variants([Ser34][Asn100][Ala105][Thr136], SNAT; [Tyr34][Asn100][Ala105][Thr136],YNAT; [Trp34][Asn100][Asp105][Thr136]; WNDT[Val34][Asn100][Asn105][Ser136], VNNS) and a Halo tag having the aminoacid sequence of SEQ ID NO: 28 (Halo-Sortase), were cloned into abacterial expression vector pET21a or pET24d. A Halo-Sortase having theamino acid sequence of SEQ ID NO: 29, which comprises a SrtA variantderived from Staphylococcus aureus (SEQ ID NO: 27) and a Halo tag (SEQID NO: 28), is used in the following examples.

1.4 Purification of Halo-Sortase

Halo-Sortase is expressed in E. coli BL21(DE3), purified and stored in5%-10% glycerol at −80° C. His-Sortase with a His₆ tag and GB1-Sortasewith a GB1 tag are prepared in a similar manner for comparison purposes.

1.5 Activity of Halo-Sortase

Procedures:

(1) Mix the purified antibody T-LCCT_(L)-HC with the linker-payloadintermediate having the structure of formula (IV-1-6) or (IV-1-6′) at anoptimal molar ratio (Ab:linker-payload intermediate=1:1-1:100) in theconjugation buffer.(2) Incubate Halo-Sortase, His-Sortase or GB1-Sortase prepared as inExample 1.4 with the mixture from step (1) at 4-40° C. for 0.5-20 h,respectively.(3) Store the product from step (2) at 4° C. or −80° C.(4) Subject the product to 12% SDS-PAGE electrophoresis to determine theconjugation efficiency.

The result is shown in FIG. 2 , the conjugation efficiencies ofHalo-Sortase, GB1-Sortase and His-Sortase are over 90%.

Example 2: Preparation of Immobilized Halo-Sortase 2.1 Preparation ofthe Chloroalkyl-Linker Modified Resin (Chloro Resin)

Methods of preparing Chloro Resin have been described in, for example,in U.S. Pat. Nos. 7,429,472, 7,888,086 and 8,202,700, which areincorporated by reference herein in their entirety. Resins used forChloro Resin preparation are shown in Table 2.

TABLE 2 Resin Code 1 NHS-activated Bestaresin 4FF HX17091 (Highlycrosslinked agarose, Bestchrom) 2 CNBr-activated Bestaresin HX17092(Highly crosslinked agarose, Bestchrom) 3 Epoxy-activated resin HX17093(Polymethyl methacrylate, Nano-Micro) 4 Epoxy-activated Bestaresin 4FFHX17094 (Highly crosslinked agarose, Bestchrom)

Procedures:

(1) Pre-treatment For NHS-Activated Resin (Bestchrom) and CNBr-ActivatedResin (Bestchrom):

Filter resin in isopropyl alcohol and wash the filter cake once withDMF, and dry by suctioning. Transfer the filter cake to a flask usingDMF and stir. Subsequently, add ethylenediamine to the mixture, and stirfor 10 to 15 h. Filter and wash the filter cake with DMF. Then drain theliquid.

For Epoxy-Activated Resins:

Filter resin in isopropyl alcohol and wash the filter cake once withH2O, and dry by suctioning. Transfer the filter cake to a flask andstir. Subsequently, add 25% to 28% concentrated ammonia water to themixture, slowly heat the system to 40-50° C. and react at 40-50° C. withstirring. Lower the temperature of the system to 20-30° C., and filterthe mixture. Wash the filter cake with H₂O until the filtrate pH reachesabout 7-8. Subsequently, wash the filter cake with DMF and drain theliquid.

(2) Transfer the filter cake from the step (1) to a flask and stir.Sequentially add DMF containing chloroalkyl substrate having thestructure of formula (I-1-1) as described above and triethylamine to thesystem. React with stirring. Subsequently, filter the system and washthe filter cake with DMF and finally drain the liquid.(3) Transfer the filter cake from step (2) to a flask. Turn on thestirring. Sequentially add Ac2O and triethylamine to the mixture. Reactwith stirring. Then, filter the mixture and wash the filter cake withDMF. Then wash the filter cake with H₂O and drain the liquid. Finally,transfer the mixture to a container using 20% ethanol for storage.

Results: the Chloro Resin having the structure of formula (II-1) isobtained:

wherein the

is highly crosslinked agarose resin or polymethyl methacrylate resin.

2.2 Immobilization of Halo-Sortase to the Chloro Resin

Procedures:

(1) Incubate the purified Halo-Sortase prepared as in Example 1.4 andthe Chloro resin prepared as in Example 2.1 at room temperature for 10min-24 h;(2) Wash the resin with 20 mM Tris-HCl, 150 mM NaCl (pH 6.0-10.0) forthree times;(3) Determine the enzymatic activity of the immobilized Halo-Sortase;(4) Optionally, pack the column with the immobilized Halo-Sortase toobtain a Halo-Sortase column;(5) Wash the immobilized Halo-Sortase from step (3) or the Halo-Sortasecolumn from step (4) with 20 mM Tris-HCl, 1-3 M NaCl (pH 6.0-10.0),0.1-1.0 M NaOH and store at 4° C.

Example 3: Characterization of the Chloro Resin

Procedures:

(1) Take 250 μl of each Chloro Resin to be tested, add in an excessiveamount of Halo-Sortase, place the tubes on a rotor, and incubate for 2 hat room temperature;(2) At different time points (15 min, 30 min, 1 h and 2 h, respectively)of the immobilization reaction, take a drop of supernatant of eachChloro Resin by centrifuging the tube at 3000 g for 3 min at roomtemperature, and determine the concentration of Halo-Sortase in thesupernatant using a Nanodrop spectrophotometer;(3) Calculate the concentration of Halo-Sortase at each time point,which is then subtracted from the initial concentration of Halo-Sortaseto obtain the amount of immobilized Halo-Sortase at each time point, andplot a curve showing the amount of Halo-Sortase immobilized on theChloro Resin as a function of the conjugation time.

The results are shown in FIG. 3 : the amount of Halo-Sortase immobilizedon the Chloro Resin reaches to a plateau at the time point 2 h, showingthe maximum capacity of each Chloro Resin.

Example 4: Characterization of Immobilized Halo-Sortase

Procedures:

(1) Immobilize a fixed amount of Halo-sortase onto the Chloro Resin asin Example 2.2.(2) Wash the immobilized enzyme with 5-10 resin volumes of 1× StorageBuffer for three times, each time centrifuging at 3000 g for 3 min atroom temperature to pellet the immobilized enzyme resin; resuspend theimmobilized enzyme in conjugation buffer.(3) Take 25 μl of each immobilized enzyme resin, add in 200 μl of GFPprotein comprising a donor recognition motif LPETGG and small moleculereaction buffer containing the linker-payload intermediate, whichcomprises an acceptor recognition motif GGG coupled to a small moleculecompound, to start the conjugation reaction.(4) At different time points (15 min, 30 min, 1 h and 2 h, respectively)of the conjugation reaction, take a sample of the supernatant forHIC-HPLC analysis.(5) Determine the conjugation efficiency by HIC-HPLC. The results areshown in FIG. 4 (the conjugation activity is expressed as DAR overtime).

Example 5: Solubility of the ADC Products Catalyzed by Halo-Sortase atLow Temperature

Procedures:

(1) Prepare ADC samples using recombinant sortase proteins GB1-Sortase,His-Sortase or Halo-Sortase according to the method described in the“General procedures”.(2) Let the ADC samples stand on ice for 10 min.(3) Centrifuge the ADC samples at 12000 g for 5 min and transfer thesupernatant to a new tube.(4) Add 20 μl 1×SDS Loading Buffer to the precipitate of step (3) todissolve the precipitate and take 5 μl for sample loading.(5) Boil all the samples at 95° C. for 10 min and load the samples to a12% SDS-PAGE gel for analysis.

The results are shown in FIG. 5 : most of the GB1-Sortase catalyzed ADCsprecipitate when placed on ice, while there is no precipitation fromHalo-Sortase or His-Sortase catalyzed ADCs. The results suggest thatADCs catalzyed by Halo-Sortase or His-Sortase are more stable under coldconditions comparing to those catalyzed by GB1-Sortase and may survivein a conjugation process with a longer hold-time, which is typical forbioconjugate production. Therefore, His-Sortase and Halo-Sortase have anadvantage over GB1-Sortase in terms of product solubility.

Example 6: Separation of Halo-Sortase and ADC 6.1 Separation ofHalo-Sortase and ADC Using Anion Exchange Chromatography (AEX)

Procedures:

(1) Pack column with Q Sepharose FF medium.(2) Equilibrate column with 20 min Tris-HCl (pH 7.5) and apply thesamples (A: ADCs, B: Halo-Sortase, and C: Mixture of ADCs andHalo-Sortase (the mass ratio of ADC:Halo-Sortase is about 100:1,respectively).(3) Flush the column with 20 min Tris-HCl (pH 7.5) until the baseline,eluent pH, and conductivity are stable.(4) Regenerate the column with 20 min Tris-HCl, 1 M NaCl (pH 7.5).(5) Perform cleaning-in-place (CIP) with 1 M NaOH.

The results are shown in FIG. 6 : at pH 7.5, ADCs pass through the Q FFcolumn(flow-through [FT] mode), which is consistent with theconventional ADC&Ab purification procedure; Halo-Sortase binds to the QFF column (bind/elute [B/E] mode) and is eluted by 20 min Tris-HCl, 1 MNaCl pH 7.5; for the mixture of ADCs & Halo-Sortase, ADCs pass throughthe Q FF column, while Halo-Sortase binds the Q FF column, and the twoare well-separated.

6.2 Separation of Halo-Sortase and ADC Using Cation ExchangeChromatography (CEX)

Procedures:

(1) Pack column with Capto S ImpAct medium.(2) Equilibrate column with 20 min citric acid-sodium citrate (pH 6.2)and apply the samples (A: ADCs and B: Halo-Sortase, and C: Mixture ofADCs and Halo-Sortase, respectively).(3) Equilibrate column with 20 min citric acid/sodium citrate (pH 6.2)until the baseline, eluent pH, and conductivity are stable.(4) Elute the sample with 20 min citric acid/sodium citrate, 160 minNaCl, pH 6.2.(5) Regenerate the column with 20 min citric acid/sodium citrate, 1 MNaCl, pH 6.2 and perform cleaning-in-place (CIP) with 1 M NaOH.

The results are shown in FIG. 7 : at pH 6.2, ADCs bind to the Capto Simpact column (bind/elute [B/E] mode); at pH 7.5, Halo-Sortase flowsthrough the Capto S impact column (flow-through [FT] mode); for themixture of ADCs & Halo-Sortase, ADCs bind to the Capto S impact column,while Halo-Sortase passes through, and the two are well-separated.

6.3 Comparison on the Removal of His₆-Sortase or Halo-Sortase from theReaction Product

The isoelectric points of His₆-Sortase and Halo-Sortase and modes ofchromatography (AEX and CEX) used for separating His₆-Sortase orHalo-Sortase and the reaction product ADC are shown in Table 3.His₆-Sortase has an isoelectric point of 8.92, which is close to that ofADC (8-9). His₆-Sortase and ADC both bind to the cation exchanger in CEXand both pass through the anion exchanger in AEX, making it difficult toseparate the two. Halo-Sortase has an isoelectric point of 5.7.Halo-Sortase and ADC can be easily separated using either AEX or CEX.

TABLE 3 His₆-Sortase Halo-Sortase ADC Isoelectric point 8.92 5.70 8-9Mode on AEX Flow-though Bind-and-elute Flowt-hough Mode on CEXBind-and-elute Flow-though Bind-and-elute

Example 7: Preparation of ADCs Using Antibody Purified by the Protein AAffinity Chromatography (Process 2) 7.1 Conjugation Reaction

The target ADCs are prepared according to the method described in the“General procedures”, using the monoclonal antibody (mAb) obtained fromProtein A affinity chromatography of the HCCF (i.e., mAb eluate fromProtein A affinity chromatography). The content of HCP is 1000 to 2000ppm in the antibody feed. The container used for conjugation reaction isthe Halo-Sortase column prepared in Example 2.2. The crude conjugatemixture is collected as the flow-through from the Halo-Sortase column.According to the HIC-HPLC analysis (see FIG. 9 ), the DAR of ADCsprepared is 1.83. The conjugation efficiency is 91.6% (Table 4).

TABLE 4 DAR0 DAR1 DAR2 Conjugation Area (%) Area (%) Area (%) DAREfficiency 0.80 15.31 83.89 1.83 91.6%

7.2 Removal of the Impurities

The crude conjugate mixture collected in 7.1 is subjected to AEX and CEXsequentially; and the samples containing the target ADCs from each step(ADC flow-through from AEX and ADC eluate from CEX, respectively) arecollected. The mAb eluate from Protein A affinity chromatography, ADCflow-through from AEX and ADC eluate from CEX are analyzed using ELISAand qPCR to determine the amount of residual impurities.

An approximately one-log reduction (90%) of the HCP content is observedafter the AEX chromatography. And the levels of HCP, DNA and Protein Aare all reduced to below 1 ppm after the series of chromatographypurification (see FIG. 10 ).

Example 8: Preparation of ADCs Using the Antibody Obtained from HCCF(Process 1) 8.1 Conjugation Reaction

The target ADCs are prepared according to the method described in the“General procedures”, using HCCF as the antibody feed. The content ofCHO HCP is 100000 to 1000000 ppm in the antibody feed.

The container used for conjugation reaction is the Halo-Sortase columnprepared in Example 2.2. The crude conjugate mixture is collected as theflow-through from the Halo-Sortase column. According to the HIC-HPLCanalysis (see FIG. 11 ), the DAR of ADCs prepared is 1.81, and theconjugation efficiency is 90.5% (see Table 5).

TABLE 5 DAR0 DAR1 DAR2 Conjugation Area (%) Area (%) Area (%) DAREfficiency 0.33 18.51 81.16 1.81 90.5%

8.2 Detection and Removal of the Impurities

The crude conjugate mixture collected in 8.1 is subjected to Protein Aaffinity chromatography, AEX and CEX sequentially; and theADC-containing solutions from each step (ADC eluate from Protein Aaffinity chromatography, ADC flow-through from AEX and ADC eluate fromCEX, respectively) are analyzed using ELISA and qPCR to determine theamount of residual impurities.

An approximately 80% reduction of the HCP content and an approximatelythree-log reduction (99.9%) of the DNA content are observed after theAEX chromatography. An approximately 93% reduction of the HCP content isobserved after the CEX chromatography. And the levels of DNA and ProteinA are reduced to below 5 ppm, the level of HCP is reduced to below 40ppm, after the series of chromatography purification (see FIG. 11 ).

The process of the present disclosure is suitable for fast preparationof ADCs, especially at small scales, for example in a lab, or in thehigh through-put preparation of ADCs for the purposes such asinvestigation for bioactivities.

Example 9: Preparation of ADCs Using Antibody Purified by the Protein AAffinity Chromatography (Process 3) 9.1 Conjugation Reaction

The target ADCs are prepared according to the method described in the“General procedures”, using the monoclonal antibody obtained from theProtein A affinity chromatography of the HCCF (i.e., mAb eluate fromProtein A affinity chromatography). The container used for conjugationreaction is the Halo-Sortase column prepared in Example 2.2. The crudeconjugate mixture is collected as the flow-through from the Halo-Sortasecolumn.

9.2 Removal of the Impurities

The crude conjugate mixture collected in 9.1 is subjected to Protein Aaffinity chromatography, AEX and CEX sequentially, in a manner similarto 8.2. The mAb eluate from Protein A affinity chromatography, ADCeluate from Protein A affinity chromatography,

ADC flow-through from AEX and ADC eluate from CEX are analyzed usingELISA and qPCR to determine the amount of residual impurities. Theresult shows that the levels of HCP and Protein A are reduced to below 2ppm after the series of chromatography purification (see FIG. 12 ).Therefore, applying two Protein A affinity chromatography steps wouldnot influence the quality of the product in the aspect of possiblyleached Protein A. And the low levels of impurities in the eluate of thesecond Protein A affinity chromatography step indicates that this methodcould tolerate higher loading amount/higher complexity of the loadingsample. And thus the present example can be interpreted as a conceptualvalidation of purification process which is more complicated as comparedto those described in Example 7 and Example 8.

9.3 Removal of Residual Enzyme Contaminants (i.e., Enzymes that Fall Offfrom the Halo-Sortase Column after Conjugation)

Crude conjugate mixture is collected as the flow-through from theHalo-Sortase column as described in Example 9.1 and then subjected toProtein A affinity chromatography, AEX and CEX sequentially. The crudeconjugate mixture, ADC eluate from Protein A affinity chromatography,ADC flow-through from AEX and ADC eluate from CEX are subjected to ELISAanalysis to determine the amount of residual enzyme contaminants

As shown in FIG. 13 , a three-log reduction (99.9%) of residual enzymecontaminants is achieved after a series of chromatography purification.In particular, comparing to one step purification using Protein Aaffinity chromatography only, anion exchange chromatography and cationexchange chromatography further reduce the amount of residual enzyme inthe final products by about 78%.

Example 10: Separation of the Linker-Payload Intermediate and ADC

Conventional process for the removal of the linker-payload intermediatefrom the crude conjugate mixture containing the target ADCs typicallyinvolves ultrafiltration (UF). However, the shear force produced duringthe ultrafiltration process increases the risk of protein moleculeaggregation. This is of particular concern in the case of removing smallmolecules (e.g., the linker-payload intermediate) from the product ADCs,because of the long ultrafiltration time and the enhanced hydrophobicityof ADCs as compared to the unconjugated protein molecules.

The present disclosure provides several choices of unit operations(process steps) to remove the linker-payload intermediate. Examples ofthe chromatography steps are provided hereinafter. In these examples,the ADC samples are prepared according to the method described in the“General procedures”, using the antibody obtained from the Protein Aaffinity chromatography of the HCCF, or use HCCF as the antibody feed.The chromatography steps are performed according to the methodsdescribed in the “General procedures”.

10.1 Protein A Affinity Chromatography

The target ADCs are prepared using a method similar to 9.1. The crudeconjugate mixture is collected as the flow-through from the Halo-Sortasecolumn and then subjected to Protein A affinity chromatography usingProtein A chromatography media provided by different suppliers (Biomaxand GE). The method employing GE Protein A comprises a Wash step. Andthe method employing Biomax Protein A does not comprise a Wash step. Theresults are shown in FIG. 14 .

10.2 CEX

The target ADCs are prepared using a method similar to 9.1. The crudeconjugate mixture is collected as the flow-through from the Halo-Sortasecolumn and then subjected to CEX chromatography using CEX media providedby GE.

The result is shown in FIG. 14 .

10.3 Protein A Affinity Chromatography, AEX and CEX

The target ADCs are prepared using a method similar to 7.1. The crudeconjugate mixture is collected as the flow-through from the Halo-Sortasecolumn and then subjected to Protein A affinity chromatography, AEX andCEX sequentially. The samples containing the target ADCs from each stepis analyzed by RP-HPLC for determination of residual linker-payloadintermediate (Linker-Toxin).

A more than four-log reduction (more than 99.99%) of the linker-payloadintermediate content is observed after the Protein A affinitychromatography.

The present disclosure provides diversified methods for the removal ofthe linker-payload intermediate, for example Protein A affinitychromatography, AEX, CEX and a combination thereof. Further purificationcan also be achieved by additional steps such as ultrafiltration and/ordiafiltration, after the polishing step. Therefore, the linker-payloadintermediate could be thoroughly removed.

Sequence Listing (sortase A) SEQ ID NO: 1AKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATE VK (sortase A) SEQ ID NO: 2KTPEIPKDKSKMAGYIKVPDAEIEEPVYPGPATPE QLNRGVSFAEGNESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSD VKVLDEHKGEKNQLTLITCDNYNKETGVWEKRKIFVAKEIK (sortase A) SEQ ID NO: 3 KAPAIPKDKSKMAGYIKVPDAEIEEPVYPGPATPEQLNRGVSFAEGNESLTDQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKVGNETRKYKMTSIRNVDPSDVKVLDEHKGEKNQLTLITCDNYNKNTGVWEKRKIF VAKQIN (sortase A) SEQ ID NO: 4KTPTIPKDKSKMAGYIEVPDAEIKEPVYPGPATLE QLNRGVSFAEGDESLDQQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNQTRKYKMTKIHDVNPSD VEVLDEQKGKKNQLTLITCDDYNEKTGVWEKRKIFIATQVN (sortase A) SEQ ID NO: 5 KAVEIPKDKSKMAGYIKIPDAEIEEPVYPGPATPEQLNRGVSFAEGNESLSDQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSDVKVLDEHKGEKNQLTLITCDNYNKETGVWEKRKIF VAKEIK (sortase A) SEQ ID NO: 6KAPEIPKDKSKMAGYIKVPDAEIEEPVYPGPATPE QLNRGVSFAEGNESLTDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSD VKVLDEHKGEKNQLTLITCDNYNKNTGVWEKRKIFVAKQIN (sortase A) SEQ ID NO: 7 KKPTIPKDKSKMAGYIEVPDAEIKEPVYPGPATPEQLNRGVSFAEGDESLDQQNIAIAGHTFTDRPHYQF TNLKAAKKGSKVYFKVGNEVRKYKMTKIHDVDPTEVKVLDEHKGKKNQLTLITCDDYNEQTGVWEKRKIF VATQVN (sortase A) SEQ ID NO: 8KAVEIPKDKSKMVGYIKVPDAEIEEPVYPGPATPE QLNRGVSFAEGNESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSD VKVLDEHKGEKNQLTLITCDNYNKETGVWEKRKIFVAKEIK (sortase A) SEQ ID NO: 9 ESPQIPKDKAKMAGYIEIPDAQIKEPVYPGPATPQQLNRGVSFAEGDESLNQQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKVGNQTRKYKITKIHDVKPTEVKVLDEHPSKKNQLTLITCDDYNEQTGVWETRKIF VATQMN (sortase A) SEQ ID NO: 10STPKIPSDKSKMAGYIEVPDAQIKEPVYPGPATPE QLNRGVSFAEGDESLNQQNISIAGHTFTDRPHYQFTNLKSAKIGSKVYFKTGNQTRKYKITKIRDVKPTE VKVLDEHPNKKNQLTLITCDDYNEETGVWETRKIFIATQIN (sortase A) SEQ ID NO: 11 ERPQIPKDKAKMAGYIEIPDAQIKEPVYPGPATPQQLNRGVSFAEGDESLYQQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKVRNQTRKYKITKIHDVKPTEVKVLDEHPSKKNQLTLITCDDYNEQTGVWETRKIF VATQMN (sortase A) SEQ ID NO: 12STPKIPSDKSKMAGYIEVPDAQIKEPVYPGPATPE QLNRGVSFAEGDESLNQQNISIAGHTFTDRSHYQFTNLKSAKIGSKVYFKTGNQTRKYKITKIRDVKPTE VKVLDEHPNKKNQLTLITCDDYNEETGVWETRKIFIATQIN (sortase A) SEQ ID NO: 13 EKPTISKDKSKMTGYISIPDADIKEPVYPGPATPEQLNRGVSFAEEDESLDDQNISIAGHTFIDRPNYQF TNLKAAKKGSKVTFKIGNETRKYKMTSIRDVNPEDVEVLDEHKGKKNQLTLITCDDYNENTGVWEKRKIF VAEEVK (sortase A) SEQ ID NO: 14EKPTISKDKSKMTGYISIPDADIKEPVYPGPATPE QLNRGVSFAEEDESLDDQNISIAGHTFTDRPNYQFTNLKAAKKGSKVTFKIGNETRKYKMTSIRDVDPDA VEVLDENKGKKNQLTLITCDDYNENTGVWEKRKIFVAEQIK (sortase A) SEQ ID NO: 15 EKPTISKDKSKMTGYISIPDADIKEPVYPGPATPEQLNRGVSFAEEDESLDDQNISIAGHTFTDRPNYQF TNLKAAKKGSKVTFKTGNETRKYKMTSIRDVDPDAVEVLDENKGKKNQLTLITCDDYNENTGVWEKRKIF VAEQIK (sortase A) SEQ ID NO: 16ETPTIPKDKSKMAGYISIPDAEIKEPVYPGPATPE QLNRGVSFAEEDEKLDDQNISIAGHTFIDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVNPDD VKVLDEHKGETNQLTLITCDNYNEQTGIWEKRKIFVAKQIN (sortase A) SEQ ID NO: 17 ETPTIPKDKSKMAGYISIPDAEIKEPVYPGPATPEQLDRGVSFAEEDEKLDDQNISIAGHTFIDRPHYQF TNLKAAKKGSKVYFKVGNETRKYKMTSIRDVNPDDVKVLDEHKGETNQLTLITCDNYNEQTGIWEKRKIF IAKQIN (sortase A) SEQ ID NO: 18EKPTIPKDKSKMAGYISVPDAEIKEPVYPGPATPE QLNRGVSFAEGDESLDDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGDETREYKMTSIRDVDPED VQVLDEHKGETNQLTLITCDNYNQQTGVWEKRKIFVAKQIK (sortase A) SEQ ID NO: 19 ERPTIPKNKSEMAGYISIPDAEIKEPVYPGPATLEQLNRGVSFAEGDESLDDQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKVGDETREYKMTSIRDVDPEDVQVLDEHKGETNQLTLITCDNYNQQTGVWEKRKIF VAKQIK (sortase A) SEQ ID NO: 20QTPTIPKDKSKMAGYISVPDAEIKEPVYPGPATPE QLNRGVSFAEEDESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDQ VEVLNEHKGEKNQLTLITCDDYNEQTGVWEKRKIFVAKQVK (sortase A) SEQ ID NO: 21 QTPTIPKDKTKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFAEKDESLSDQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDEVEVLDEHKGEKNQLTLITCDDYNEQTGVWEKRKIF VAKQVK (sortase A) SEQ ID NO: 22ERPTIPKDKSKMAGYISVPDAEIKEPVYPGPATLE QLNRGVSFAEGDESLDDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGDETREYKMTSIRDVNPED VQVLDEHEGETNQLTLITCDNYNQQTGVWEKRKIFVAKQIK (sortase A) SEQ ID NO: 23 QTPTIPKDKTKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFAEKDESLSDQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDEVEVLDEHKGEKNQLTLITCDDYNEQTGVWEKRKIF VAKQVN (sortase A) SEQ ID NO: 24DKPTIPKDKAEMAGYLRIPDADINEPVYPGPATPE QLNRGVSFAEEQESLDDQNIAIAGHTYIGRPHYQFTNLKAAKKGSKVYFKVGNETREYKMTTIRDVNPDE IDVLDEHRGDKNRLTLITCDDYNEKTGVWEKRKIFIAEQIK (sortase A) SEQ ID NO: 25 QTPTIPKDKTKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFTEKDESLSDQNISIAGHTFTDRPHYQF TNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDEVEVLDEHKGEKNQLTLITCDDYNEQTGVWEKRKIF VAKQVN (sortase A) SEQ ID NO: 26DKPTIPKDKAEMAGYLRIPDADINEPVYPGPATPE QLNRGVSFAEEQESLDDQNIAIAGHTYIGRPHYQFTNLKAAKKGSKVYFKVGNETREYKMTTIRDVDPDE IDVLDEHRGDKNRLTLITCDDYNEKTGVWEKRKIFIAEQIK (sortase A, SNAT counterpart of SEQ ID NO: 1) SEQ ID NO: 27AKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSE QLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTA VGVLDEQKGKDKQLTLITCDDYNEKTGVWETRKIFVATEVK (Halo tag) SEQ ID NO: 28 MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMG KSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDE WPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELP IAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPD LIGSEIARWLSTLEISG (Halo-Sortase)SEQ ID NO: 29 MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMG KSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDE WPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELP IAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPD LIGSEIARWLSTLEISGGGGGSGGGGSAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSF AEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVGVLDEQK GKDKQLTLITCDDYNEKTGVWETRKIFVATEVK

1-54. (canceled)
 55. An immobilized ligase, comprising a ligase fusionprotein immobilized to a support, wherein the ligase fusion protein hasan isoelectric point (pI) that is lower than that of the ligase fromwhich the ligase fusion protein is derived.
 56. The immobilized ligaseaccording to claim 55, wherein the support comprises a haloalkyl linkersuch that the ligase fusion protein is immobilized on the supportthrough covalent interaction between the haloalkyl linker and the Halotag.
 57. The immobilized ligase according to claim 56, wherein thehaloalkane linker is a chloroalkane linker produced by a chloroalkylsubstrate having the structure of formula (I-1):

wherein u is an integer of 1 to 20, v is an integer of 0 to 20, and w isan integer of 1 to
 19. 58. The immobilized ligase according to claim 57,wherein the support has the structure of formula (II):

wherein u is an integer of 1 to 20, v is an integer of 0 to 20, and w isan integer of 1 to 19;

 is a resin, a bead, a membrane, a gel, a matrix, a film, a plate, awell, a tube, a glass slide or a surface.
 59. The immobilized ligaseaccording to claim 55, wherein the ligase is a sortase A.
 60. Theimmobilized ligase according to claim 59, wherein the sortase Acomprises a modified amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-26 or an amino acid sequence having asequence identity of at least about 85% thereto, and wherein themodification comprises amino acid substitutions selected from SNAT (Ser,Asn, Ala, Thr), YNAT (Tyr, Asn, Ala, Thr), WNDT (Trp, Asn, Asp, Thr) andVNNS (Val, Asn, Asn, Ser) at positions 34, 100, 105 and
 136. 61. Theimmobilized ligase according to claim 60, wherein the modified aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-26 oran amino acid sequence having a sequence identity of at least about 95%thereto.
 62. The immobilized ligase according to claim 60, wherein thesortase A comprises the amino acid sequence of SEQ ID NO: 27 or an aminoacid sequence having a sequence identity of at least about 85% thereto.63. The immobilized ligase according to claim 62, wherein the sortase Acomprises the amino acid sequence of SEQ ID NO: 27 or an amino acidsequence having a sequence identity of at least about 95% thereto. 64.The immobilized ligase according to claim 55, wherein the Halo tagcomprises the amino acid sequence of SEQ ID NO: 28 or an amino acidsequence having a sequence identity of at least about 85% thereto. 65.The immobilized ligase according to claim 64, wherein the Halo tagcomprises the amino acid sequence of SEQ ID NO: 28 or an amino acidsequence having a sequence identity of at least about 95% thereto. 66.The immobilized ligase according to claim 55, wherein the ligase has anisoelectric point (pI) of about 7.5 to about 10.0, the Halo tag has anisoelectric point of about 4.5 to about 5.0, and the pI of the ligasefusion protein is about 2.0 to about 4.5 pH units lower than that of theligase.
 67. The immobilized ligase according to claim 58, wherein

is a resin.
 68. The immobilized ligase according to claim 67, whereinthe resin is an agarose resin, a silicone resin, a polymethylmethacrylate resin or cellulose resin.
 69. The immobilized ligaseaccording to claim 68, wherein the resin is a highly crosslinked agaroseresin.